The

metabolic

significance

of

octulose

phosphates

in

the

photosynthetic

carbon

reduction

cycle

in

spinach

octulose

phosphates

were

formed

during

photosynthetic

fixation

and

were

measured

in

spinach

leaves

and

chloroplasts

Because

mono

and

bisphosphates

of

d-glycero-d-ido-octulose

are

the

active

ketosugar

intermediates

of

the

L-type

pentose

pathway

it

was

proposed

that

they

may

also

be

reactants

in

a

modified

Calvin

Benson

Bassham

pathway

reaction

scheme

This

investigation

therefore

initially

focussed

only

on

the

ido-epimer

of

the

octulose

phosphates

even

though

d-glycero-d-altro-octulose

mono

and

bisphosphates

were

also

identified

in

chloroplasts

and

leaves

predominantly

labelled

positions

and

of

d-glycero-d-ido-octulose

consistent

with

labelling

predictions

of

the

modified

scheme

The

kinetics

of

incorporation

into

ido-octulose

was

similar

to

its

incorporation

into

some

traditional

intermediates

of

the

path

of

carbon

while

subsequent

exposure

to

rapidly

displaced

the

isotope

label

from

octulose

with

the

same

kinetics

of

label

loss

as

some

of

the

confirmed

Calvin

pathway

intermediates

This

is

consistent

with

octulose

phosphates

having

the

role

of

cyclic

intermediates

rather

than

synthesized

storage

products

-LRB-

Storage

products

do

n't

rapidly

exchange

isotopically

labelled

carbons

with

unlabelled

-RRB-

Introduction

The

formulation

of

the

Calvin

Benson

Bassham

pathway

hereafter

called

Calvin

cycle

of

photosynthesis

-LRB-

PS

-RRB-

was

heavily

dependent

on

the

elucidation

of

a

reaction

sequence

for

the

pentose

pathway

-LRB-

PP

-RRB-

of

glucose

metabolism

in

the

biochemistry

of

tissues

A

reaction

sequence

for

the

PP

was

proposed

from

the

results

of

experiments

in

which

liver

pea

leaf

and

pea

root

enzyme

preparations

were

used

to

catalyse

the

conversion

of

-LSB-

-RSB-

Ribose

-LRB-

Rib

-RRB-

hexose

and

unlabelled

glyceraldehyde

-LRB-

Gap

-RRB-

which

were

formed

in

vitro

over

a

period

-LRB-

Horecker

et

al.

-RRB-

For

many

years

Williams

and

co-workers

investigated

and

published

results

of

research

on

PP

in

liver

some

neoplasms

adipose

tissue

heart

colonocytes

and

photosynthetic

tissues

-LRB-

see

Williams

et

al.

Williams

for

published

listings

-RRB-

These

investigations

showed

that

the

conclusion

drawn

from

the

results

of

the

fundamental

experiments

on

which

the

PP

reaction

sequence

is

based

-LRB-

Horecker

et

al.

Gibbs

and

Horecker

-RRB-

was

erroneous

and

because

of

its

duration

the

results

were

incapable

of

being

interpreted

due

to

the

random

scattering

of

isotope

in

the

glucose

-LRB-

Glc

-RRB-

product

The

labelling

pattern

of

Glc

was

the

result

of

a

prediction

labelling

experiment

that

was

used

to

hypothesize

a

reaction

sequence

for

the

PP

The

unpredictable

and

randomized

isotope

scattering

of

in

Glc

was

finally

shown

to

be

due

to

the

activity

of

the

extensive

rates

versus

the

slower

rates

of

mass

transfer

reactions

catalysed

by

the

group

transferring

enzymes

aldolase

-LRB-

EC

-RRB-

-LRB-

Ald

-RRB-

transketolase

-LRB-

EC

-RRB-

-LRB-

TK

-RRB-

and

transaldolase

-LRB-

EC

-RRB-

-LRB-

TA

-RRB-

-LRB-

Flanigan

et

al.

-RRB-

Williams

et

al.

-LRB-

-RRB-

demonstrated

that

the

reversible

interconversions

of

Rib

and

hexose

and

triose

phosphates

by

liver

and

the

other

tissues

mentioned

above

also

involved

ido

and

altro-octulose

mono

and

bisphosphates

sedoheptulose

-LRB-

Seh

-RRB-

and

a

low

concentration

of

arabinose

-LRB-

Ara

-RRB-

as

intermediates

together

with

a

proposal

for

the

inclusion

of

three

new

enzyme

activities

in

the

PP

reaction

sequence

-LRB-

see

Fig.

-RRB-

Fig.

L-Type

PP

of

glucose

metabolism

The

pathway

features

reactions

of

arabinose

d-glycero-d-ido-octulose

and

sedoheptulose

mono

and

bisphosphates

together

with

new

enzymes

to

catalyse

their

reactions

-LRB-

Williams

and

Clark

Williams

et

al.

Williams

-RRB-

Calvin

and

colleagues

based

the

reaction

sequence

which

regenerated

pentose

phosphates

-LRB-

especially

Ru

-RRB-

from

hexose

and

triose

phosphates

in

PS

on

a

selection

of

the

reactions

of

a

reverse-acting

classical

PP

-LRB-

Calvin

-RRB-

thereby

providing

important

credibility

at

the

time

for

the

PP

which

still

carried

an

author-imposed

tentative

caveat

It

was

later

noted

-LRB-

Clark

et

al.

-RRB-

that

the

omission

of

the

above

new

PP

intermediates

and

enzymes

from

the

Calvin

scheme

provided

further

explanations

of

some

of

the

early

criticisms

and

anomalies

of

the

Calvin

pathway

scheme

-LRB-

Clark

et

al.

Stiller

Kandler

and

Gibbs

Beck

and

Hopf

-RRB-

The

investigations

described

here

were

made

in

order

to

-LRB-

a

-RRB-

search

for

evidence

that

d-g-d-i-oct

phosphates

may

be

formed

by

carbon

fixation

reactions

in

spinach

during

PS

and

-LRB-

b

-RRB-

quantitatively

measure

any

formation

of

octulose

phosphates

and

investigate

whether

their

proposed

path

of

synthesis

conformed

with

a

theoretical

extension

of

the

photosynthetic

reaction

scheme

shown

in

Fig.

This

paper

reports

the

results

of

studies

on

the

formation

and

functions

of

octulose

phosphates

with

a

particular

initial

focus

on

d-glycero-d-ido-octulose

and

the

together

with

other

novel

intermediates

in

spinach

leaves

and

chloroplasts.Fig

initial

hypothesis

for

a

revised

reaction

scheme

for

the

reductive

PP

of

photosynthetic

carbon

reduction

in

plants

The

hypothesis

differs

from

the

Calvin

Pathway

by

the

inclusion

of

new

enzymes

and

reactions

by

octulose

and

arabinose

phosphates

and

is

tested

by

the

results

reported

in

this

paper

The

figure

shows

the

distributions

-LRB-

for

one

cycle

-RRB-

of

the

coloured

carbon

of

into

traditional

and

proposed

new

intermediates

of

a

modified

Calvin

Pathway

The

initial

singular

attention

on

the

ido

epimer

rather

than

an

inquiry

that

also

included

d-glycero-d-altro-octulose

phosphates

rested

on

the

exclusive

roles

assigned

to

d-g-d-i-oct

phosphates

in

the

L-type

PP

-LRB-

Fig.

-RRB-

However

a

final

conclusion

drawn

from

the

study

suggests

that

both

epimeric

forms

of

octulose

phosphates

occur

and

that

both

may

have

roles

in

the

path

of

carbon

in

PS

The

investigation

used

the

same

tracer

and

other

methodologies

that

were

pioneered

by

Calvin

and

his

team

in

the

unravelling

of

a

path

of

carbon

in

PS

The

accompanying

paper

reports

the

results

of

a

subsequent

mass

spectrometric

investigation

of

the

incorporation

of

into

to

sugar

phosphates

in

spinach

chloroplasts

during

light-driven

PS

Materials

and

methods

Growth

of

plant

material

Spinach

-LRB-

Spinacia

oleracea

L.

var

Yates

-RRB-

was

grown

by

water

culture

in

a

glasshouse

under

natural

lighting

according

to

established

methods

-LRB-

Anderson

and

Boardman

-RRB-

Depending

on

the

season

leaves

were

harvested

five

or

eight

weeks

after

sowing

the

seeds

Chemical

and

chromatographic

materials

Enzymes

and

cofactors

for

analysis

and

synthesis

of

sugar

phosphates

were

obtained

from

either

Sigma

Chemical

Co.

St.

Louis

MO

USA

or

from

Boehringer-Mannheim

Corp.

Mannheim

Germany

and

compounds

were

from

Amersham

-LRB-

Bucks

England

-RRB-

Other

analytical

reagent

grade

chemicals

were

obtained

from

Ajax

Chemicals

Sydney

Australia

BDH

Poole

UK

or

E.

Merck

Darmstad

Germany

Chromatographic

media

were

obtained

from

the

following

sources

Ion

exchange

resins

from

BioRad

Richmond

CA

U.S.A.

Phenylboronate

agarose

gel

-LRB-

Matrex

PBA

-RRB-

from

Amicon

Corp.

Danvers

MA

USA

Thin

layer

chromatography

plates

from

E.Merck

paper

from

Whatman

Maidstone

UK

Sephadex

and

Sephadex

from

Pharmacia

Uppsala

Sweden

Chromatographic

columns

were

from

Pharmacia

or

LKB

Bromma

Sweden

Ultrafiltration

apparatus

and

Diaflow

membranes

were

obtained

from

Amicon

Lexington

MA

USA

Extraction

and

analysis

of

photosynthetic

tissue

metabolites

by

gas-liquid

chromatography

Young

spinach

leaves

-LRB-

weeks

-RRB-

were

detached

from

plants

which

had

been

exposed

to

full

sunlight

for

h

and

then

placed

in

liquid

nitrogen

The

tissue

was

ground

to

a

fine

powder

using

a

stainless

steel

mortar

and

pestle

and

perchloric

acid

was

added

The

temperature

of

this

mixture

was

raised

to

°C

and

the

solid

was

blended

with

the

acid

using

a

Potter-Elvejhem

homogenizer

The

liquid

was

separated

from

insoluble

matter

by

centrifugation

at

g

for

min

The

pellet

was

re-extracted

with

perchloric

acid

and

the

two

extracts

combined

The

solution

was

neutralized

with

M

KOH

and

stored

at

°C

for

several

hours

to

complete

the

precipitation

of

potassium

perchlorate

which

was

then

removed

by

centrifugation

at

g

for

min

The

supernatant

fluid

was

treated

with

acid-washed

activated

carbon

-LRB-

Norite

-RRB-

to

remove

nucleotides

and

aromatic

compounds

and

was

lyophilized

The

extract

was

dissolved

in

ml

of

M

acetate

buffer

pH

and

units

of

acid

phosphatase

-LRB-

EC

from

potato

-RRB-

added

Mannitol

-LRB-

μmol

-RRB-

and

erythritol

-LRB-

μmol

-RRB-

were

added

as

internal

standards

The

mixture

was

incubated

at

°C

to

achieve

complete

sugar

phosphate

hydrolysis

The

free

sugars

were

recovered

by

thermally

denaturing

the

protein

followed

by

centrifugation

and

treatment

of

the

supernatant

fraction

with

a

three-fold

excess

-LRB-

calculated

from

the

amount

of

buffer

used

-RRB-

of

mixed

bed

resin

-LRB-

Dowex

mesh

H

form

Dowex

mesh

form

-RRB-

The

completeness

of

deonization

was

established

using

a

Radiometer

conductivity

meter

The

solution

was

lyophilized

and

stored

at

°C

Lyophilized

extracts

were

converted

to

their

TMS

ethers

immediately

before

analysis

using

the

procedure

of

Williams

et

al.

-LRB-

-RRB-

Dimethylformamide

and

bis-trimethylsilyltrifluoroacetamide

-LRB-

μl

of

each

-RRB-

were

added

and

the

mixture

stirred

for

min

at

°C

The

analyses

were

carried

out

using

a

Pye-Unicam

gas

chromatograph

-LRB-

series

-RRB-

fitted

with

two

glass

columns

-LRB-

cm

cm

-RRB-

packed

with

on

mesh

Supelco

Bellefonte

PA

USA

FID

responses

were

recorded

on

a

computing

integrator

-LRB-

Spectra-Physics

model

-RRB-

Photosynthesis

experiments

with

whole

spinach

leaves

isotope

tracer

and

pulse

chase

investigations

with

whole

spinach

leaves

were

conducted

using

an

exact

application

of

the

methods

and

apparatus

described

by

Hatch

and

Slack

-LRB-

-RRB-

This

procedure

and

the

method

for

the

ethanol

extraction

preparation

of

the

soluble

fraction

and

isolation

of

sugar

phosphates

was

described

by

Bartlett

et

al.

-LRB-

-RRB-

Sugar

phosphates

were

separated

by

formate

anion-exchange

chromatography

and

further

purified

using

two-dimensional

TLC

or

paper

chromatography

and

the

solvents

-LRB-

Kapuscinski

et

al.

-RRB-

The

insoluble

residue

remaining

after

ethanol

extraction

of

all

sugar

phosphates

was

extensively

washed

with

water

ethanol

and

acetone

The

amount

of

radioactivity

in

the

insoluble

glucan

was

determined

using

liquid

scintillation

counting

-LRB-

Arora

et

al.

-RRB-

Preparation

of

whole

leaf

homogenate

Approximately

g

of

fresh

spinach

leaves

were

washed

briefly

in

distilled

water

de-ribbed

and

cut

into

small

segments

to

facilitate

maceration

The

material

was

homogenized

in

ml

of

mM

Tris

HCl

buffer

-LRB-

pH

-RRB-

containing

mM

EDTA

and

strained

through

two

layers

of

Miracloth

-LRB-

Calbiochem

-RRB-

After

centrifugation

at

g

for

min

the

preparation

was

concentrated

four-fold

by

ultrafiltration

using

a

MW

cut-off

membrane

-LRB-

Amicon

-RRB-

The

concentrated

solution

was

dialysed

for

h

against

six

changes

of

the

homogenization

buffer

All

operations

were

carried

out

at

°C

Chloroplast

isolation

The

procedure

for

the

isolation

of

intact

chloroplasts

is

based

on

a

method

which

involves

a

two-step

gradient

of

the

silica-sol

Percoll

-LRB-

Pharmacia

Biotech

Sweden

-RRB-

-LRB-

Robinson

-RRB-

Five

to

six-weeks-old

spinach

leaves

were

harvested

in

the

early

morning

in

order

to

avoid

the

accumulation

of

starch

granules

which

can

rupture

the

chloroplast

envelope

during

centrifugation

The

amount

of

leaf

material

required

to

produce

a

satisfactory

yield

of

chloroplasts

was

not

critical

but

the

yield

depended

on

the

quality

of

the

leaves

and

increased

with

the

amount

of

material

used

The

harvested

leaves

were

de-ribbed

and

floated

in

basins

of

water

Immediately

before

isolating

the

chloroplasts

the

leaves

were

illuminated

for

min

with

incandescent

light

at

an

intensity

of

μ

E

m

s

During

this

period

crushed

ice

was

added

to

the

water

to

maintain

the

temperature

at

°C

Typically

g

of

coarsely

chopped

leaves

were

homogenized

in

ml

of

medium

for

s

using

a

Polytron

blender

-LRB-

setting

-RRB-

fitted

with

a

probe

The

homogenizing

medium

at

pH

was

chilled

to

a

semi-frozen

consistency

before

use

and

contained

mM

sorbitol

mM

mM

mM

isoascorbic

acid

and

bovine

serum

album

-LRB-

BSA

-RRB-

-LRB-

Jensen

and

Bassham

-RRB-

The

brei

was

filtered

through

a

double

layer

of

Miracloth

-LRB-

Calbiochem

-RRB-

The

filtrate

was

centrifuged

at

g

for

min

in

a

Sorvall

refrigerated

centrifuge

-LRB-

DuPont

Medical

Products

Newtown

CT

USA

-RRB-

using

either

an

or

rotor

The

supernatant

fluid

was

discarded

and

the

sedimented

material

was

gently

resuspended

using

a

fine

soft

brush

in

ml

of

a

medium

at

pH

containing

mM

sorbitol

mM

HEPES

KOH

mM

EDTA

mM

and

mM

-LRB-

Jensen

and

Bassham

-RRB-

An

aliquot

-LRB-

ml

-RRB-

of

the

suspension

was

then

placed

into

each

of

two

Corex

tubes

and

carefully

underlaid

with

ml

of

Percoll

medium

made

to

with

resuspension

medium

and

centrifuged

Finally

the

chloroplasts

were

suspended

in

the

resuspension

medium

-LRB-

ml

-RRB-

and

stored

on

ice

The

above

procedures

were

carried

out

on

ice

using

previously

chilled

equipment

All

parameters

-LRB-

density

yield

photosynthetic

activity

etc.

-RRB-

describing

chloroplast

properties

were

related

to

the

Chl

content

measured

according

to

the

following

procedure

Twenty

five

to

g

of

leaves

produced

between

mg

and

mg

of

Chl

Provided

the

quality

of

leaf

material

was

satisfactory

and

the

Percoll

was

fresh

the

method

unfailingly

produced

a

maximum

yield

of

highly

active

chloroplasts

which

were

better

than

intact

Typical

examples

of

preparations

had

the

following

mean

values

and

S.D.

Activity

-LRB-

n

-RRB-

μmol

h

mg

Chl

Intactness

-LRB-

n

-RRB-

Chlorophyll

assay

The

Chl

content

of

isolated

chloroplast

suspensions

was

estimated

by

the

method

of

Arnon

-LRB-

-RRB-

In

whole

leaf

experiments

Chl

was

estimated

using

the

method

of

Vernon

-LRB-

-RRB-

Measurements

of

chloroplast

polarographic

activity

and

intactness

The

activity

of

the

isolated

chloroplast

suspensions

was

determined

polarographically

using

a

Clark

oxygen

electrode

The

reaction

mixture

-LRB-

ml

-RRB-

contained

mM

sorbitol

mM

EDTA

mM

mM

mM

HEPES

mM

Pi

mM

pyrophosphate

units

of

catalase

-LRB-

EC

-RRB-

mM

and

chloroplast

suspension

equivalent

to

μg

of

Chl

The

evolution

of

isolated

chloroplast

suspensions

illuminated

in

an

assay

medium

at

°C

was

recorded

using

a

Goerz

Metrawatt

chart

recorder

and

the

rate

of

oxygen

evolution

was

calculated

from

the

linear

portion

of

the

trace

The

chloroplasts

were

illuminated

at

an

intensity

of

μE

m

s

using

the

quartz-halogen

light

from

a

slide

projector

In

the

experiments

described

here

the

range

of

acceptable

activity

of

chloroplast

preparations

is

μmol

h

mg

Chl

Light

measurements

were

made

using

a

Hansatech

Quantum

Sensor

-LRB-

Hansatech

Instruments

King

's

Lynn

Norfolk

JL.UK

-RRB-

The

intactness

of

chloroplasts

was

measured

by

the

ferricyanide

reduction

method

-LRB-

Lilley

et

al.

-RRB-

Isotopic

tracer

studies

with

isolated

chloroplasts

Experiments

were

carried

out

in

the

oxygen

electrode

-LRB-

unless

stated

otherwise

-RRB-

by

the

procedure

described

for

the

assay

of

chloroplast

activity

-LRB-

μCi

-RRB-

was

introduced

when

the

rate

of

oxygen

evolution

reached

a

linear

rate

The

reaction

was

terminated

by

rapid

transfer

of

the

mixture

to

boiling

ethanol

-LRB-

w/v

-RRB-

The

insoluble

material

was

removed

by

centrifugation

and

washed

once

with

a

small

volume

of

water

The

ethanol

was

evaporated

from

the

combined

supernatant

solutions

in

a

rotary

evaporator

After

passage

through

a

cation-exchange

column

-LRB-

Bartlett

et

al.

-RRB-

the

sugar

phosphates

were

separated

and

isolated

by

anion-exchange

column

chromatography

and

further

purified

by

two-dimensional

TLC

or

paper

chromatography

-LRB-

Kapuscinski

et

al.

-RRB-

The

isolation

of

stromal

metabolites

used

the

method

of

Heldt

-LRB-

-RRB-

which

relied

on

rapid

separation

of

intact

chloroplasts

from

a

reaction

medium

using

the

following

silicon

oil

method

Fifty

μl

of

M

formic

acid

and

μl

of

silicone

oil

mixture

-LRB-

and

mixed

in

a

ratio

of

-RRB-

were

placed

in

a

microfuge

tube

followed

by

μl

of

a

chloroplast

suspension

that

had

been

preincubated

with

-LRB-

specific

radioactivity

mCi

mmol

-RRB-

The

extracts

of

the

pellets

containing

the

aqueous

fractions

that

were

pooled

from

four

tubes

were

lyophilized

and

the

sugar

phosphates

therein

were

isolated

by

anion-exchange

column

chromatography

as

described

in

Methods

Sugar

phosphate

peaks

isolated

in

the

column

eluates

were

identified

and

quantitatively

measured

using

the

specific

enzymatic

and

colourimetric

procedures

of

Williams

et

al.

-LRB-

-RRB-

and

Kapuscinski

et

al.

-LRB-

-RRB-

Preparation

of

stromal

enzyme

extracts

-LRB-

SEP

-RRB-

from

spinach

chloroplasts

The

soluble

-LRB-

stromal

-RRB-

enzyme

fraction

was

prepared

essentially

as

described

by

Furbank

and

Lilley

-LRB-

-RRB-

for

pea

chloroplasts

Intact

spinach

chloroplasts

were

isolated

with

assayed

rates

of

evolution

in

excess

of

μmol

h

mg

Chl

Typically

a

chloroplast

suspension

equivalent

to

mg

of

Chl

was

pelleted

by

centrifugation

at

g

for

min

in

an

Sorvall

rotor

at

°C

The

supernatant

solution

was

removed

and

ml

of

chilled

mM

HEPES

buffer

pH

containing

mM

DTT

was

added

The

pellet

was

resuspended

by

gentle

shaking

The

centrifuge

tube

was

kept

on

ice

for

approximately

min

after

which

it

was

centrifuged

for

min

at

g

in

a

refrigerated

Sorvall

centrifuge

to

separate

the

soluble

proteins

from

the

membrane

fraction

The

clear

supernatant

fluid

representing

the

stromal

fraction

of

the

chloroplasts

was

concentrated

to

ml

in

an

Amicon

ultrafiltration

cell

using

a

membrane

-LRB-

nominal

MW

cut-off

-RRB-

Finally

dialysis

was

carried

out

against

ml

of

dialysis

buffer

containing

mM

HEPES

KOH

-LRB-

pH

-RRB-

mM

DTT

mM

and

mM

KCl

This

last

step

was

completed

in

h

The

pelleted

membranes

were

retained

for

Chl

analysis

The

activity

of

the

extracts

was

stable

for

h

from

completion

of

the

dialysis

step

Some

experiments

used

an

alternative

stromal

isolation

procedure

based

on

the

method

of

Kaiser

and

Bassham

-LRB-

-RRB-

The

chloroplast

suspension

was

pelleted

as

described

above

but

lysis

of

organelles

was

achieved

with

mM

HEPES

NaOH

mM

mM

EDTA

-LRB-

pH

-RRB-

and

the

same

buffer

was

used

for

dialysis

Assay

of

the

reductive

PP

using

stromal

enzyme

preparation

-LRB-

SEP

-RRB-

The

activity

of

the

reductive

PP

in

the

stromal

extracts

was

determined

by

the

method

of

Furbank

and

Lilley

-LRB-

-RRB-

The

progress

of

the

reaction

was

followed

spectrophotometrically

using

light

path

cuvettes

The

assay

mixture

contained

mM

HEPES

KOH

buffer

-LRB-

pH

-RRB-

mM

mM

KCl

mM

NADH

mM

EDTA

mM

DTT

mM

ATP

mM

creatine

phosphate

mM

units

of

creatine

kinase

-LRB-

EC

-RRB-

units

of

glyceraldehyde-phosphate

dehydrogenase

-LRB-

EC

-RRB-

units

of

phosphoglycerate

kinase

-LRB-

EC

-RRB-

SEP

equivalent

to

μg

of

Chl

was

added

to

make

a

final

volume

of

μl

SEP

was

preincubated

for

min

at

°C

in

mM

mM

DTT

mM

before

addition

of

the

test

sugar

phosphate

substrate

Usually

the

following

test

substrates

were

individually

used

at

mM

concentration

in

the

reaction

mixture

Rib

dihydroxyacetonephosphate

-LRB-

DHAP

-RRB-

or

Seh

Maximum

catalytic

capacity

of

the

non-oxidative

segment

of

the

PP

This

was

determined

by

following

the

conversion

of

ribose

to

hexose

in

a

coupled

enzyme

assay

as

described

by

Williams

et

al.

-LRB-

-RRB-

The

reaction

mixture

contained

mM

TEA

HCl

buffer

pH

mM

NADP

mM

units

of

phosphoglucose

isomerase

-LRB-

EC

-RRB-

-LRB-

PGI

-RRB-

units

of

glucose

dehydrogenase

-LRB-

EC

-RRB-

-LRB-

Glc

-RRB-

mM

ribose

and

stromal

extract

equivalent

to

μg

of

Chl

in

a

total

volume

of

ml

Synthesis

of

isotopically

labelled

compounds

-LSB-

-RSB-

Arabinose

-LSB-

-RSB-

d-g-d-a-oct

and

-LSB-

-RSB-

d-g-d-i-oct

were

prepared

by

the

method

of

Arora

et

al.

-LRB-

-RRB-

-LSB-

-RSB-

Sedoheptulose

was

prepared

by

phosphorylation

of

sedoheptulose

-LRB-

altro-heptulose

-RRB-

with

-LSB-

-RSB-

ATP

using

rabbit

muscle

phosphofructokinase

-LRB-

EC

-RRB-

-LRB-

PFK

-RRB-

The

reaction

mixture

-LRB-

ml

-RRB-

contained

μmol

Tris

HCl

buffer

-LRB-

pH

-RRB-

μmol

of

μmol

of

-LSB-

-RSB-

ATP

-LRB-

μCi

-RRB-

μmol

of

Seh

and

units

of

PFK

The

mixture

was

incubated

at

°C

and

was

complete

in

h

The

title

compound

was

purified

using

formate-anion

exchange

chromatography

and

TLC

as

described

above

and

was

shown

by

enzymatic

analysis

to

be

free

of

Fru

Unlabelled

d-glycero-d-ido

and

d-glycero-d-altro-octulose

and

esters

were

prepared

as

described

in

Kapuscinski

et

al.

-LRB-

-RRB-

Measurements

of

phosphotransferase

activity

Arora

et

al.

-LRB-

-RRB-

published

three

assay

procedures

for

the

minimum

estimate

of

the

activity

of

d-glycero-d-ido-octulose

or

d-glycero-d-altro-octulose

d-altro-heptulose

-LRB-

PT

-RRB-

Two

of

these

assays

measured

the

reaction

for

the

non-oxidative

PP

-LRB-

Fig.

-RRB-

in

the

direction

of

octulose

formation

The

third

procedure

and

the

one

detailed

here

measured

the

activity

in

the

flux

direction

of

the

RPP

-LRB-

see

Fig.

and

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

-RRB-

Only

the

third

method

was

effective

when

SEP

was

the

crude

source

of

the

putative

enzyme

activity

It

is

a

radiochromatographic

discontinuous-stop

assay

and

is

applied

as

follows

The

reaction

mixture

in

a

volume

of

ml

contained

μmol

of

-LSB-

-RSB-

octulose

-LRB-

μCi

μmol

-RRB-

μmol

Seh

μmol

of

TEA

HCl

buffer

-LRB-

pH

-RRB-

and

μmol

of

KCl

The

mixture

was

incubated

for

min

at

°C

and

was

initiated

by

the

addition

of

SEP

equivalent

to

μg

of

Chl

Aliquots

-LRB-

ml

-RRB-

were

sampled

at

min

intervals

during

the

time-course

of

the

reaction

and

injected

into

an

equal

volume

of

perchloric

acid

Denatured

protein

was

removed

by

centrifugation

the

pH

of

the

supernatant

fluid

adjusted

to

with

KOH

and

precipitated

separated

by

centrifugation

The

activity

of

the

enzyme

was

estimated

by

separating

the

labelled

octulose

mono

and

bis-phosphates

by

ion-exchange

chromatography

as

follows

A

precisely

known

volume

of

the

above

supernatant

fraction

was

applied

onto

a

column

-LRB-

mm

mm

-RRB-

of

ion-exchange

resin

-LRB-

AG

mesh

formate

form

-RRB-

followed

by

ml

of

deionized

water

The

sugar

phosphates

were

eluted

by

successive

washes

with

M

HCOOH

-LRB-

approx.

ml

-RRB-

which

quantitatively

removed

monophosphate

esters

and

ml

of

M

HCOOH

M

solution

which

eluted

sugar

bisphosphates

Fractions

-LRB-

ml

-RRB-

of

the

eluted

sugar

phosphates

at

each

time

point

were

analysed

for

radioactivity

and

the

amount

of

octulose

bisphosphate

formed

was

calculated

from

the

integrated

peaks

of

radioactivity

Confirmation

of

the

identity

and

amount

of

radioactivity

in

the

octulose

phosphates

used

two-dimensional

radiochromatography

of

each

of

the

above

fractions

and

the

solvents

-LRB-

Kapuscinski

et

al.

-RRB-

Control

experiments

using

only

a

single

substrate

accompanied

each

activity

measurement

Arabinose

activity

with

SEP

The

reaction

mixture

-LRB-

ml

-RRB-

containing

μmol

TEA

HCl

buffer

-LRB-

pH

-RRB-

μmol

Rib

μmol

of

-LSB-

-RSB-

Ara

-LRB-

μCi

-RRB-

and

SEP

equivalent

to

μg

of

Chl

was

incubated

for

h

at

°C

The

reaction

was

terminated

by

heating

and

all

sugar

phosphates

were

isolated

with

greater

than

recovery

of

isotope

An

identical

control

reaction

using

boiled

SEP

accompanied

all

incubations

Processed

samples

were

chromatographed

on

paper

using

either

the

or

PBA

solvents

and

the

chromatograms

exposed

to

X-ray

film

-LRB-

Kodak

X-O

mat

RP

-RRB-

-LRB-

Bleakley

et

al.

-RRB-

Positional

isotopic

analysis

of

sugar

phosphates

Following

PS

experiments

with

leaves

or

chloroplasts

Glc

Rib

d-g-d-i-oct

and

PGA

were

isolated

resolved

by

ion-exchange

chromatography

and

purified

by

two-dimensional

TLC

or

paper

chromatography

The

fraction

containing

Glc

was

concentrated

and

chromatographed

twice

on

paper

-LRB-

Whatman

-RRB-

using

the

solvent

of

Wood

-LRB-

-RRB-

followed

by

solvent

A

of

Bandurski

and

Axelrod

-LRB-

-RRB-

The

Glc

was

located

by

autoradiography

and

comparison

with

a

stained

standard

marker

compound

-LRB-

Rosenberg

-RRB-

and

was

then

eluted

from

the

paper

with

deionized

water

The

above

regime

ensured

that

the

isolated

Glc

was

free

of

any

other

hexose

phosphates

The

phosphate

group

of

Glc

was

removed

by

enzymatic

hydrolysis

as

follows

Solid

Glc

was

dissolved

in

ml

of

mM

glycine

NaOH

buffer

pH

and

units

of

Alkaline

phosphatase

-LRB-

EC

-RRB-

from

Escherichia

coli

was

added

and

the

mixture

incubated

at

°C

The

progress

of

the

reaction

was

monitored

by

assaying

the

release

of

inorganic

phosphate

using

the

method

of

Tashima

and

Yoshimura

-LRB-

-RRB-

When

the

reaction

was

completed

the

mixture

was

poured

into

a

suspension

of

mixed-bed

resin

-LRB-

meq

of

Dowex

H

form

and

meq

of

Bio

x

form

-RRB-

which

was

stirred

for

approx.

min

The

solution

was

filtered

through

a

sintered

glass

funnel

-LRB-

porosity

-RRB-

and

the

residual

cake

of

resin

thoroughly

washed

with

deionized

water

The

glucose

filtrate

was

freeze-dried

and

stored

at

°C

until

used

The

percentage

distribution

of

isotope

in

the

individual

carbons

of

glucose

was

determined

as

using

a

combination

of

microbiological

and

chemical

methods

-LRB-

Williams

et

al.

-RRB-

octulose

phosphates

from

spinach

leaves

or

chloroplast

extracts

were

isolated

and

purified

as

above

and

degraded

using

the

methods

of

Williams

et

al.

-LRB-

-RRB-

Following

isolation

by

anion-exchange

and

TLC

chromatography

Rib

and

Ara

were

dephosphorylated

-LRB-

Williams

et

al.

-RRB-

and

Ara

and

Rib

were

chemically

degraded

using

the

periodate

method

-LRB-

Genovese

et

al.

-RRB-

-LSB-

-RSB-

acid

derived

from

DHAP

was

chemically

degraded

by

the

procedure

of

Andrews

et

al.

-LRB-

-RRB-

Recovery

of

as

and

measurement

of

radioactivity

Discs

-LRB-

cm

-RRB-

of

Whatman

No.

filter

paper

were

used

to

collect

barium

carbonate

precipitates

The

discs

were

washed

with

water

absolute

ethanol

and

diethyl

ether

dried

and

weighed

before

use

was

deposited

on

the

papers

using

gentle

suction

then

washed

with

water

alcohol

and

ether

The

discs

were

dried

under

vacuum

over

silica

and

equilibrated

at

room

temperature

before

weighing

on

a

semi-micro

balance

Approx.

mg

of

dried

was

exactly

weighed

placed

into

a

vial

with

ml

of

scintillation

cocktail

and

counted

for

radioactivity

as

a

suspension

in

the

Cab-O-Sil

-LRB-

Godfrey

L.

Cabot

Inc.

-RRB-

scintillant

of

Cluley

-LRB-

-RRB-

Replication

and

statistical

analysis

Data

presented

are

the

mean

values

-LRB-

S.D.

-RRB-

from

results

of

five

or

more

experiments

or

mean

values

-LRB-

P.E.

-RRB-

-LRB-

Probable

Error

-RRB-

for

all

experiments

conducted

on

different

days

Results

and

discussion

GLC

analysis

of

perchloric

acid

extracts

of

spinach

leaves

Principal

aims

of

the

experiments

were

an

investigation

of

the

occurrence

identity

and

quantitative

levels

of

the

ido

and

altro-octuloses

in

fresh

photosynthetic

tissue

and

the

assessment

of

their

hypothesized

functions

-LRB-

especially

that

of

d-g-d-i-oct

-RRB-

in

PS

It

was

noted

that

Charlson

and

Richtmyer

-LRB-

-RRB-

and

Begbie

and

Richtmyer

-LRB-

-RRB-

had

isolated

and

characterized

octulose

from

avocado

and

Primula

officinalis

respectively

In

a

remarkable

finding

Howarth

et

al.

-LRB-

-RRB-

showed

that

d-glycero-d-ido-octulose

represented

of

the

total

carbohydrates

in

fully

hydrated

leaves

and

of

dried

leaf

matter

of

the

resurrection

plant

Craterostigma

plantagineum

Heath

-LRB-

-RRB-

also

showed

evidence

for

the

L-type

but

not

the

F-type

PP

in

Chlorella

sorokiniana

Since

the

mono

and

bisphosphate

esters

of

d-g-d-i-oct

are

a

unique

feature

of

the

L-type

PP

-LRB-

Fig.

-RRB-

the

need

to

identify

and

measure

these

esters

in

fresh

spinach

leaf

and

chloroplasts

is

obvious

Different

methods

were

used

to

establish

whether

d-glycero-d-ido-octulose

occurred

in

spinach

leaves

The

first

approach

used

the

simple

and

sensitive

GLC

method

A

gas

chromatogram

of

the

pertrimethylsilyl

-LRB-

TMSi

-RRB-

derivatives

of

the

dephosphorylated

sugar

phosphates

extracted

from

spinach

leaves

is

shown

in

Fig.

Fig.

liquid

chromatogram

of

dephosphorylated

TMSi-derivatized

sugars

extracted

from

young

spinach

leaves

during

light-driven

carbon

fixation

Octuloses

and

arabinose

feature

in

the

chromatogram

For

details

see

Materials

and

methods

section

The

high

molecular

weight

region

was

well

resolved

and

allowed

tentative

identification

-LRB-

by

retention

times

and

co-chromatography

with

authentic

compounds

-RRB-

of

d-g-d-i-octulose

and

its

altro-epimer

d-g-d-a-oct

Estimation

of

the

amounts

of

these

compounds

from

the

known

response

factors

of

the

gas

chromatograph

gave

values

of

nmol

mg

Chl

for

ido-octulose

and

nmol

mg

Chl

for

altro-octulose

These

values

are

tentative

because

other

compounds

may

co-elute

with

the

octuloses

e.g.

the

TMS

ethers

of

heptitols

have

similar

retention

times

to

those

of

eight

carbon

sugars

In

addition

the

GLC

method

used

in

this

study

can

not

differentiate

between

free

and

phosphorylated

sugars

Different

approaches

were

therefore

adopted

to

determine

the

levels

of

specifically

phosphorylated

octuloses

present

in

photosynthesizing

tissue

isolation

from

spinach

leaves

fixation

and

pulse

chase

studies

The

second

approach

used

the

combination

of

formate

ion-exchange

chromatography

followed

by

phenylboronic

acid

and

two-dimensional

TLC

to

achieve

isolation

identification

and

complete

separation

of

d-g-d-i-oct

from

all

other

sugar

phosphates

-LRB-

see

Methods

-RRB-

This

methodology

both

confirmed

and

extended

the

GLC

findings

and

unequivocally

demonstrated

that

d-g-d-i-oct

occurred

in

spinach

leaf

extracts

The

conditions

adopted

for

the

study

of

the

time-course

of

fixation

into

the

products

of

PS

involved

an

exact

application

of

the

method

of

Hatch

and

Slack

-LRB-

-RRB-

This

provided

steady-state

conditions

for

PS

in

intact

spinach

leaves

at

a

concentration

of

ppm

that

followed

from

a

ml

injection

of

mCi

of

and

light

intensity

of

direct

sunlight

-LRB-

μE

m

s

-RRB-

The

rate

of

incorporation

into

the

soluble

leaf

fraction

-LRB-

an

index

of

the

total

fixed

-RRB-

was

approximately

linear

-LRB-

cpm

s

mg

Chl

-RRB-

during

a

interval

-LRB-

Fig.

panel

A

-RRB-

The

labelling

of

PGA

was

marginally

more

rapid

and

extensive

than

that

of

Glc

which

reached

isotope

saturation

in

the

interval

s.Fig

the

kinetics

of

isotope

incorporation

from

into

a

selection

of

sugar

phosphates

in

whole

spinach

leaves

during

light-driven

PS

for

s

The

time

course

of

the

total

fixed

in

the

soluble

fraction

of

the

leaf

extract

is

also

shown

Labelled

sugar

phosphates

were

resolved

by

anion-exchange

chromatography

and

were

then

individually

isolated

and

further

purified

by

two-dimensional

paper

or

TLC

chromatography

as

described

in

the

Materials

and

methods

Section

The

incorporated

in

glucose

and

PGA

-LRB-

panel

A

-RRB-

was

an

order

of

magnitude

greater

than

that

incorporated

in

the

sugar

phosphates

shown

in

panel

B.

Panel

A

Total

isotope

fixed

radioactivity

incorporated

in

Glc

and

PGA

Panel

B.

Rib

other

pentose

Seh

Δ

and

d-g-d-i-oct

Glc

was

labelled

at

an

approx

rate

of

cpm

s

mg

Chl

-LRB-

Fig.

Panel

A

-RRB-

The

radioactivity

in

PGA

and

Glc

was

an

order

of

magnitude

greater

than

that

fixed

in

all

other

sugar

phosphates

investigated

-LRB-

see

Fig.

panel

B

-RRB-

The

fast

rate

of

incorporation

into

Rib

was

initially

inclined

to

linear

for

s

-LRB-

cpm

mg

Chl

-RRB-

The

bisphosphates

Seh

and

d-g-d-i-oct

were

more

slowly

labelled

at

and

cpm

s

mg

Chl

respectively

The

kinetics

and

relative

degree

of

isotope

incorporation

into

individual

PS

intermediates

shown

at

Fig.

are

very

similar

to

the

results

for

incorporation

in

Scenedesmus

-LRB-

Benson

et

al.

-RRB-

Moreover

d-g-d-i-oct

was

labelled

early

and

was

saturated

with

isotope

at

s

at

which

point

its

concentration

in

the

leaf

was

nmol

mg

Chl

and

the

rate

of

label

incorporation

was

of

that

fixed

in

the

soluble

fraction

When

the

above

experiments

were

conducted

in

the

dark

the

distribution

of

in

the

leaf

soluble

fraction

was

uneven

and

at

s

was

only

of

that

measured

in

the

light

of

the

sugar

phosphates

shown

at

the

A

and

B

panels

of

Fig.

were

dark

labelled

Hatch

and

Slack

-LRB-

-RRB-

described

an

apparatus

for

measurements

of

the

kinetics

of

fixation

and

exchange

by

pulse

chase

in

bulk

samples

of

intact

leaves

undergoing

PS

Their

method

was

able

to

delineate

Calvin

Cycle

intermediates

which

rapidly

exchanged

for

during

and

following

a

short

pulse

with

thereby

identifying

rapidly

cycling

compounds

These

are

clearly

distinguished

by

their

delabelling

kinetics

-LRB-

Figs.

and

-RRB-

from

the

cytoplasmic

storage

compounds

sucrose

starch

and

cationic

compounds

that

retained

or

increased

their

levels

of

following

the

pulse

phase

-LRB-

Fig.

-RRB-

Fig.

the

results

of

pulse

chase

experiments

where

spinach

leaves

were

confined

in

for

s

-LRB-

the

pulse

-RRB-

and

were

then

exposed

to

non-radioactive

air

-LRB-

the

chase

-RRB-

See

methods

The

figure

shows

the

time-course

of

the

partition

of

isotope

into

various

fractional

groups

of

compounds

in

spinach

leaves

following

their

exposure

to

the

chase

with

unlabelled

for

s

and

illuminated

to

the

equivalent

of

direct

sunlight

It

is

of

note

that

starch

and

cationic

compounds

retained

and

continued

to

show

increasing

radioactivity

while

the

radioactivity

in

the

total

soluble

fraction

and

the

pool

of

anionic

compounds

declined

The

time-course

of

changes

in

radioactivity

are

shown

for

starch

neutral

and

anionic

compounds

the

cationic

fraction

and

total

soluble

radioactivity

ΔFig

the

change

in

the

of

individual

sugar

phosphates

and

sucrose

in

spinach

leaves

during

the

time-course

of

the

chase

with

unlabelled

Sugar

phosphates

from

leaves

that

had

been

subjected

to

the

pulse-chase

experiments

-LRB-

See

Fig.

results

-RRB-

were

separated

by

anion-exchange

chromatography

and

individually

purified

by

two

dimensional

TLC

or

paper

chromatography

See

Materials

and

methods

for

details

Panel

A

shows

the

kinetics

over

s

of

the

decline

in

radioactivity

of

Rib

Fru

DHAP

and

d-g-d-i-oct

Panel

B

shows

the

time-course

over

s

of

the

decline

in

radioactivity

of

PGA

Glc

Fru

and

the

initial

increase

in

radioactivity

in

sucrose

Taken

together

the

results

of

Figs.

and

showed

that

d-g-d-i-oct

was

rapidly

labelled

and

saturated

with

isotope

during

fixation

It

was

delabelled

on

exposure

to

during

a

comparative

study

which

showed

that

it

exhibited

the

same

kinetics

of

loss

as

other

cycling

bisphosphate

intermediates

of

the

Calvin

Pathway

It

was

therefore

unambiguously

distinguished

from

slow

carbon

storage

and

other

cytoplasmic

end

products

of

PS

by

the

above

kinetics

d-glycero-d-ido-octulose

in

spinach

chloroplasts

The

above

results

suggested

that

d-g-d-i-oct

may

have

a

chloroplast

origin

In

order

to

test

this

proposal

PS

experiments

were

carried

out

with

isolated

spinach

chloroplasts

If

d-g-d-i-oct

is

synthesized

by

enzymes

of

the

reductive

PP

it

should

become

rapidly

labelled

following

the

metabolism

of

phosphate

-LRB-

Pi

-RRB-

or

It

was

noted

above

that

d-g-d-i-oct

incorporated

isotope

during

PS

by

isolated

chloroplasts

in

the

presence

of

-LRB-

see

Methods

-RRB-

These

observations

were

further

tested

using

as

the

isotopic

marker

-LRB-

see

Methods

section

-RRB-

in

order

to

investigate

whether

d-g-d-i-oct

was

exclusively

present

in

the

organelles

and

was

not

the

result

of

external

reactions

catalysed

by

enzymes

released

from

any

ruptured

chloroplasts

Intact

chloroplasts

were

rapidly

filtered

through

a

layer

of

silicone

oil

-LRB-

Heldt

-RRB-

and

the

metabolites

were

separated

by

ion-exchange

chromatography

-LRB-

see

Methods

section

-RRB-

The

unambiguous

identification

of

the

-LSB-

-RSB-

d-g-d-i-oct

was

made

by

rechromatographing

the

recovered

octulose

phosphate

fractions

on

paper

using

the

and

solvents

From

the

known

specific

radioactivity

of

inorganic

phosphate

in

the

reaction

mixture

and

the

absolute

amount

of

in

the

purified

d-g-d-i-oct

the

concentration

of

the

sugar

phosphate

in

the

stroma

was

estimated

Using

the

value

of

the

sorbitol

impermeable

space

of

μl

mg

Chl

-LRB-

Schafer

et

al.

-RRB-

the

concentration

of

d-g-d-i-oct

was

estimated

to

be

nmol

mg

Chl

-LRB-

μM

three

determinations

-RRB-

This

value

is

less

than

that

measured

for

ido-oct

in

the

whole

leaf

PS

study

but

is

in

the

same

concentration

range

as

that

of

some

other

Calvin

Cycle

metabolites

in

spinach

chloroplasts

-LRB-

Portis

et

al.

Heldt

et

al.

Stitt

et

al.

Petterson

and

Ryde-Petterson

-RRB-

Measurement

of

the

amount

of

the

altro-epimer

of

the

octulose

bisphosphate

in

chloroplasts

was

not

made

in

this

study

-LRB-

see

the

accompanying

paper

for

the

results

of

a

mass

spectrometric

study

of

labelled

sugar

phosphates

formed

in

spinach

chloroplasts

during

PS

-RRB-

A

carry-through

of

the

external

medium

may

affect

the

estimation

of

the

stromal

volume

as

a

sorbitol

impermeable

space

and

consequently

the

determination

of

the

molar

concentration

of

metabolites

contained

in

the

stroma

However

the

amount

of

d-g-d-i-oct

in

the

stroma

is

quoted

per

mg

of

Chl

and

is

thus

independent

of

the

volume

of

external

medium

that

may

be

carried

through

silicon

oil

with

the

organelles

The

results

of

control

experiments

using

showed

that

following

centrifugation

the

external

medium

contained

less

than

of

the

d-g-d-i-oct

found

in

the

pelleted

chloroplast

fraction

Thus

it

is

concluded

that

d-g-d-i-oct

is

probably

synthesized

and

may

react

exclusively

in

the

stromal

compartment

of

spinach

chloroplasts

Its

concentration

is

estimated

to

be

μM

using

the

above

data

and

the

following

equation

Origin

of

d-g-d-i-oct

and

distributions

of

isotope

in

a

selection

of

sugar

phosphates

isolated

from

spinach

leaves

and

chloroplasts

assimilating

in

photosynthesis

The

most

likely

biosynthetic

routes

leading

to

d-g-d-i-oct

phosphate

formation

involve

catalysis

by

the

carbon

carbon

cleavage

and

group-transferring

enzymes

of

sugar

phosphate

metabolism

namely

transketolase

-LRB-

Eq

-LRB-

-RRB-

-RRB-

aldolase

-LRB-

Eq

-LRB-

-RRB-

-RRB-

and

transaldolase

-LRB-

Eq

-LRB-

-RRB-

-RRB-

The

short-term

PS

experiments

described

in

the

Methods

and

Results

sections

were

used

to

identify

which

of

the

above

three

enzymes

may

have

operated

for

the

synthesis

of

d-g-d-i-oct

phosphate

in

spinach

leaves

and

chloroplasts

However

it

is

of

note

that

the

initial

hypothesis

-LRB-

Fig.

-RRB-

for

the

modified

reductive

PP

-LRB-

Clark

et

al.

Williams

et

al.

-RRB-

proposed

that

was

formed

by

the

reaction

of

-LRB-

Eq

-LRB-

-RRB-

-RRB-

and

by

the

PT

reaction

of

-LRB-

Eq

-LRB-

-RRB-

-RRB-

-LRB-

Arora

et

al.

-RRB-

Figure

shows

that

in

one

cycle

of

fixation

d-g-d-i-oct

is

labelled

in

carbons

and

In

order

to

test

this

aspect

of

the

mechanism

of

the

pathway

the

distributions

of

isotope

into

the

individual

carbons

of

phosphoglyceric

acid

-LRB-

PGA

-RRB-

and

d-g-d-i-oct

were

determined

in

samples

isolated

from

spinach

leaves

and

chloroplasts

during

light-driven

PS

in

The

procedures

used

-LRB-

see

Methods

section

-RRB-

allowed

each

of

the

carbons

of

Glc

and

PGA

to

be

isolated

for

the

determination

of

the

degree

of

positional

carbon

isotopic

labelling

However

only

carbons

and

of

d-g-d-i-oct

were

able

to

be

individually

isolated

for

measurement

and

carbons

and

were

collectively

measured

as

a

group

-LRB-

Williams

et

al.

-RRB-

Only

the

isotope

levels

of

carbons

and

of

were

uniquely

determined

by

the

methods

applied

in

this

study

with

carbons

and

being

estimated

as

a

group

The

data

of

Table

show

the

results

of

these

experiments

using

spinach

leaves

photosynthetically

assimilating

for

and

s

respectively

-LRB-

see

Methods

section

-RRB-

The

data

of

Table

show

similar

isotope

distributions

in

PGA

Glc

and

d-g-d-i-oct

isolated

from

intact

spinach

chloroplasts

after

s

of

PS

in

Table

of

isotope

in

sugar

phosphates

isolated

from

spinach

leaves

following

specified

intervals

of

PS

in

s

of

sGlc

FoundPredicted

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-RCB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-RCB-

-RCB-

-RCB-

-RCB-

-RCB-

-RCB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

recovery

of

conduct

of

the

experiments

is

described

in

the

Methods

section

Sugar

phosphates

were

isolated

and

purified

by

ion-exchange

and

paper

chromatographic

procedures

Results

are

mean

values

of

duplicate

degradations

except

for

all

of

the

compounds

isolated

after

s

of

fixation

which

were

degraded

in

triplicate

b

c

recovered

in

the

top

and

bottom

carbons

of

d-g-d-i-oct

of

isotope

in

sugar

phosphates

isolated

from

intact

spinach

chloroplasts

following

carbon

fixation

by

light-driven

PSaCarbonPercent

specific

sPGADHAPGlc

Found

-LRB-

Theory

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-RCB-

-RCB-

-LRB-

-RRB-

-LRB-

-RRB-

The

experiments

were

carried

in

an

oxygen

electrode

apparatus

as

described

in

the

Methods

section

Illuminated

chloroplast

suspensions

-LRB-

μg

Chl

ml

-RRB-

were

incubated

with

mM

until

a

linear

rate

of

evolution

was

attained

One

hundred

μCi

of

was

added

to

adjust

the

specific

radioactivity

of

the

substrate

to

μCi

μmol

The

subsequent

steps

are

detailed

in

the

Methods

section

The

specific

radioactivity

of

individual

carbons

of

sugar

phosphates

are

the

mean

value

of

triplicate

determinations

b

c

The

percent

recovery

of

isotope

from

the

top

and

bottom

carbon

atoms

of

d-g-d-i-oct

respectively

The

method

for

calculating

the

theoritical

bracketed

values

in

Tables

and

is

given

in

the

Appendix

Degradative

data

for

PGA

DHAP

and

Glc

following

s

of

PS

are

also

listed

Both

sets

of

results

implicate

a

TK-catalysed

reaction

-LRB-

Eq

-LRB-

-RRB-

-RRB-

as

the

most

likely

route

for

the

biosynthesis

of

d-g-d-i-oct

leading

to

the

bisphosphate

by

the

reaction

of

Eq

-LRB-

-RRB-

In

order

to

compare

the

above

TK

path

of

synthesis

with

the

distribution

pattern

that

may

be

imposed

by

the

reactions

of

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

catalysed

by

either

Aldolase

-LRB-

ALD

-RRB-

or

TA

theoretical

distributions

of

isotope

in

d-g-d-i-oct

are

also

presented

as

bracketed

values

in

Tables

and

The

Appendix

shows

the

method

for

calculating

the

theoretical

values

These

distributions

were

calculated

using

the

experimentally

measured

isotope

levels

in

Glc

-LRB-

Table

-RRB-

and

an

assumption

that

Fru

formed

in

the

reductive

pathway

is

in

chemical

and

isotopic

equilibrium

with

Glc

formed

by

the

action

of

PGI

It

is

also

assumed

that

the

distributions

in

the

mono

and

bisphosphates

of

the

d-g-d-i-octuloses

of

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

are

the

same

Equation

-LRB-

-RRB-

shows

detail

of

the

spread

of

in

each

carbon

of

d-g-d-i-oct

at

s

-LRB-

see

data

Table

-RRB-

using

the

mean

values

of

triplicate

estimations

of

isotope

in

each

of

the

six

carbons

of

Glc

and

the

assumptions

described

above

The

individual

distributions

measured

in

the

top

three

carbons

of

the

d-g-d-i-oct

sampled

at

all

times

support

the

proposal

that

d-g-d-i-oct

in

spinach

leaves

-LRB-

Table

-RRB-

and

chloroplasts

-LRB-

Table

-RRB-

may

be

formed

by

the

TK

reaction

of

Eq

-LRB-

-RRB-

However

the

leaf

concentrations

of

in

and

of

d-g-d-i-oct

in

the

and

samples

and

the

chloroplast

sample

are

greater

than

expected

from

contributions

of

and

of

hexose

phosphates

to

these

carbons

of

oct

formed

by

the

reaction

of

Eq

-LRB-

-RRB-

The

level

of

in

position

of

some

ido-octulose

samples

was

significant

and

may

be

the

result

of

a

second

cycle

of

labelling

or

action

by

a

phenomenon

that

was

first

noted

by

Van

Sumere

and

Shu

-LRB-

-RRB-

Their

results

showed

an

inversion

of

of

-LSB-

-RSB-

Rib

and

production

of

-LSB-

-RSB-

Xlu

via

the

intermediacy

of

formed

by

d-arabinitol

NAD

-LRB-

EC

-RRB-

Rephosphorylation

and

isomerization

to

form

the

isotopically

inverted

aldopentose

and

its

Aldx

catalysed

exchange

with

d-g-d-i-oct

will

specifically

label

position

Contamination

isolation

recovery

and

analytical

errors

may

be

ruled

out

since

authentically

labelled

d-g-d-i-oct

samples

subject

to

the

above

procedures

showed

that

the

error

in

analysis

of

was

However

it

is

important

to

note

that

neither

the

results

of

the

Calvin

group

the

reactions

of

the

Calvin

pathway

the

scheme

of

Fig.

nor

the

results

of

Table

show

pentose

phosphates

labelled

at

positions

other

than

carbons

and

The

scrambling

of

into

carbons

and

of

-LRB-

see

Table

for

chloroplast

data

-RRB-

by

recycling

in

the

path

of

carbon

in

PS

is

predicted

to

lead

to

some

enrichment

of

isotope

into

positions

and

of

d-g-d-i-oct

-LRB-

via

the

prior

labelling

of

Fru

from

PGA

-RRB-

However

only

heavily

labelled

-LSB-

-RSB-

PGA

was

found

after

s

PS

in

spinach

leaves

-LRB-

Table

-RRB-

After

s

of

exposure

there

was

an

almost

uniform

distribution

in

all

carbons

of

Glc

consistent

with

extensive

recycling

of

the

isotope

in

the

carbon

path

of

PS

The

results

of

the

partial

degradations

of

d-g-d-i-oct

samples

do

not

support

exclusive

paths

of

synthesis

by

the

reactions

of

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

since

both

of

these

reactions

will

very

heavily

label

carbon

of

octulose

phosphate

Because

-LSB-

-RSB-

PGA

is

the

earliest

and

most

heavily

labelled

intermediate

detected

during

fixation

in

plant

PS

and

is

the

precursor

of

-LSB-

-RSB-

DHAP

then

it

follows

that

the

aldolase

reaction

of

Eq

-LRB-

-RRB-

forms

a

predominantly

labelled

-LSB-

-RSB-

d-g-d-i-oct

The

TA

reaction

of

Eq

-LRB-

-RRB-

uses

which

will

be

most

heavily

but

unequally

labelled

-LRB-

Gibbs

Effect

-RRB-

in

positions

and

in

short

time-course

experiments

-LRB-

Tables

and

-RRB-

Thus

the

transfer

of

the

-LSB-

-RSB-

DHA-TA

moiety

from

-LSB-

-RSB-

Fru

to

Ara

in

the

reaction

of

Eq

-LRB-

-RRB-

forms

d-g-d-i-oct

which

would

have

notably

labelled

These

predictions

can

not

be

made

in

longer

term

experiments

-LRB-

much

beyond

s

in

leaves

-RRB-

because

of

the

PS-induced

spread

of

label

in

PGA

and

hexose

-LRB-

Tables

and

-RRB-

The

most

supportive

evidence

for

the

TK

reaction

in

the

synthesis

of

d-g-d-i-oct

phosphate

is

best

shown

in

the

s

fixation

in

spinach

leaves

and

s

in

spinach

chloroplasts

It

is

of

note

that

the

inability

of

the

degradative

procedures

-LRB-

see

Methods

section

-RRB-

to

provide

quantitative

individual

distributions

for

all

eight

carbons

of

the

octulose

phosphates

was

addressed

using

the

mass

spectrometric

method

-LRB-

MacLeod

et

al.

-RRB-

the

results

of

which

are

reported

in

the

accompanying

paper

The

above

quantitative

labelling

of

positions

and

of

d-g-d-i-oct

was

qualitatively

confirmed

by

Bartlett

et

al.

-LRB-

-RRB-

who

used

two-dimensional

NMR

Spectrometry

and

s

of

fixation

by

PS

with

intact

spinach

leaves

-LRB-

Hatch

and

Slack

-RRB-

to

demonstrate

formation

of

-LSB-

-RSB-

d-g-d-i-oct

Their

results

also

included

the

first

demonstration

of

Aldx

catalysis

by

showing

that

the

-LSB-

-RSB-

d-g-d-i-oct

and

-LSB-

-RSB-

Rib

both

products

of

PS

interconverted

with

-LSB-

-RSB-

d-g-d-a-oct

and

-LSB-

-RSB-

Ara

While

the

above

results

and

the

scheme

of

Fig.

support

the

proposal

that

the

TK

reaction

leading

to

Oct

involved

Glc

and

Fru

it

should

be

noted

that

TK

can

accept

various

other

substrates

-LRB-

Williams

et

al.

-RRB-

Using

the

theoretical

analysis

method

dealt

with

earlier

in

this

section

it

can

be

shown

that

Seh

serving

as

a

two-carbon

fragment

donor

will

give

the

same

labelling

result

as

that

imposed

by

Fru

On

the

other

hand

reaction

between

Glc

and

Xlu

results

in

a

different

pattern

with

and

of

the

resulting

d-g-d-i-oct

having

much

heavier

labelling

than

and

thus

this

reaction

possibility

is

excluded

Finally

another

potential

TK

donor

substrate

is

β-hydroxypyruvate

Daley

and

Bidwell

-LRB-

-RRB-

showed

that

in

its

phosphorylated

form

together

with

phosphoserine

accounted

for

a

substantial

portion

-LRB-

-RRB-

of

the

fixed

by

Phaseolus

vulgaris

leaves

during

the

first

minute

of

exposure

to

in

PS

If

P-hydroxypyruvate

is

synthesized

by

a

carboxylation

process

as

the

above

authors

imply

and

is

converted

to

hydroxypyruvate

then

reaction

of

the

latter

with

Glc

would

produce

d-g-d-i-oct

labelled

in

the

same

fashion

as

that

derived

from

Glc

and

Fru

since

the

distribution

of

in

hydroxypyruvate

resembles

that

in

carbons

in

hexose

monophosphate

However

lack

of

data

prevents

our

further

speculation

on

the

possible

role

of

hydroxypyruvate

as

a

path-of-carbon

intermediate

in

the

synthesis

of

octulose

phosphates

in

spinach

during

PS

This

section

on

octulose

phosphate

synthesis

and

reactivity

in

PS

would

be

incomplete

without

mention

of

the

epimerization

of

octulose

phosphates

-LRB-

Williams

et

al.

-RRB-

In

such

a

mechanism

d-g-d-a-oct

or

the

corresponding

bisphosphate

may

be

reversibly

generated

from

the

ido-epimeric

phosphate

esters

The

manner

of

this

epimerization

and

its

significance

in

the

proposed

modification

of

the

path

of

carbon

in

PS

is

treated

in

the

last

section

of

this

Discussion

Stromal

enzyme

preparation

and

photosynthesis

In

Furbank

and

Lilley

showed

that

reaction

sequences

of

a

complete

photosynthetic

carbon

reduction

pathway

could

be

demonstrated

using

a

SEP

-LRB-

see

Methods

section

-RRB-

from

the

chloroplasts

of

peas

By

supplying

SEP

with

ATP

NAD

-LRB-

P

-RRB-

H

and

dithiothreitol

-LRB-

DTT

-RRB-

and

operating

at

pH

the

need

for

light

was

eliminated

When

SEP

was

provided

with

a

small

priming

level

of

there

was

an

immediate

and

rapid

oxidation

of

NAD

-LRB-

P

-RRB-

H

which

was

spectrophotometrically

monitored

at

nm

The

amount

of

NAD

-LRB-

P

-RRB-

H

oxidized

was

equated

with

the

number

of

moles

of

added

During

the

period

of

rapid

NAD

-LRB-

P

-RRB-

H

oxidation

it

was

shown

in

parallel

experiments

using

that

no

carbon

fixation

took

place

However

after

this

initial

rapid

period

there

followed

an

interval

of

further

NAD

-LRB-

P

-RRB-

H

oxidation

at

a

slower

rate

which

was

linear

for

at

least

min

and

which

was

accompanied

by

fixation

It

was

assumed

that

was

fixed

by

Ru

production

for

the

ribulosebisphosphate

carboxylase

-LRB-

EC

-RRB-

reaction

which

was

supported

by

the

test

sugar-P

added

to

the

SEP

reaction

mixture

-LRB-

Table

-RRB-

Furbank

and

Lilley

hypothesized

that

in

spite

of

a

dilution

of

the

intact

enzyme

complement

of

chloroplasts

and

the

loss

of

any

attendant

structural

regulation

the

pathway

catalysed

by

SEP

is

mechanistically

similar

to

the

overall

carbon

fixation

rate

that

occurs

in

intact

organelles

although

we

suggest

that

it

may

not

be

quantitatively

equivalent

for

all

intermediates

Thus

it

was

of

interest

to

prepare

SEP

from

spinach

chloroplasts

and

investigate

its

comparative

ability

to

react

octulose

and

arabinose

phosphates

and

adopt

its

use

for

measurement

of

catalytic

activity

of

the

phosphotransferase

enzyme

and

the

rates

of

the

oxidative

and

reductive

PPs

-LRB-

see

Methods

-RRB-

Determination

of

fixation

using

SEP

and

activity

of

the

reductive

PP

with

various

sugar

phosphate

substrates

The

relative

capacity

of

SEP

to

fix

-LRB-

see

Methods

-RRB-

depended

primarily

on

the

nature

of

the

sugar-P

primer

in

the

incubation

In

Table

the

rates

of

spinach

SEP-catalysed

NADH

oxidation

in

the

presence

of

various

sugar

phosphates

are

compared

with

the

data

reported

by

Furbank

and

Lilley

-LRB-

-RRB-

using

SEP

from

peas.Table

of

reductive

PP

sequences

measured

by

NADH

oxidation

in

reactions

catalysed

by

spinach

SEP

with

a

variety

of

sugar

phosphates

as

substratesSubstrateConcentration

-LRB-

mM

-RRB-

Rate

of

NADH

oxidation

-LRB-

μmol

h

mg

Chl

-RRB-

SpinachaPeabRu

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Seh

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Fru

-LRB-

-RRB-

Fru

c

-LRB-

-RRB-

Fru

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Ara

-LRB-

-RRB-

d

d-g-d-i-oct

-LRB-

-RRB-

d-g-d-a-oct

-LRB-

-RRB-

d-g-d-i-oct

-LRB-

-RRB-

d-g-d-a-oct

-LRB-

-RRB-

a

Shows

the

results

of

this

study

Results

are

mean

values

standard

deviation

The

number

of

determinations

using

different

batches

of

SEP

are

shown

in

bracketsb

Shows

the

data

of

Furbank

and

Lilley

-LRB-

-RRB-

for

peasc

When

preparations

were

made

using

sonicated

spinach

chloroplast

suspensions

and

Fru

as

substrate

varying

-LRB-

non-reproducible

-RRB-

rates

up

to

μmol

h

mg

Chl

were

recordedd

Results

were

variable

with

different

SEP

batches

three

of

the

SEP

preparations

with

Ara

did

not

support

any

NADH

oxidation

In

general

metabolites

which

immediately

precede

the

carboxylation

reaction

in

the

scheme

of

the

reductive

PP

namely

Rib

Ru

Ru

Seh

Seh

gave

very

high

rates

of

fixation

as

indicated

by

the

oxidation

rate

of

NADH

consistent

with

their

role

in

autocatalysis

-LRB-

Walker

and

Lilley

-RRB-

However

triose

arabinose

fructose

ido

and

altro-octulose

phosphates

exhibited

low

rates

that

were

notably

below

the

maximum

rate

of

fixation

in

intact

chloroplasts

Triose-P

and

hexose-P

rates

using

pea

SEP

were

also

below

the

physiological

PS

rate

In

particular

Fru

and

Glc

-LRB-

results

not

shown

-RRB-

were

very

inefficient

substrates

for

spinach

SEP

giving

much

lower

values

than

those

reported

by

Furbank

and

Lilley

-LRB-

Table

-RRB-

for

pea

preparations

results

which

we

were

able

to

confirm

Lilley

and

Walker

-LRB-

-RRB-

also

reported

that

substrate

concentrations

of

Fru

and

a

reconstituted

chloroplast

system

yielded

very

low

rates

for

the

enablement

or

support

of

Calvin

Cycle

regenerative

activity

by

this

substrate

A

number

of

modifications

of

the

original

procedure

were

introduced

in

an

attempt

to

obtain

higher

rates

of

fixation

with

several

sugar

phosphates

Fru

in

particular

These

modifications

included

variations

in

the

composition

of

the

low

osmolality

buffer

used

for

the

disruption

of

the

organelles

freeze

thawing

and

sonication

of

chloroplasts

the

inclusion

of

various

components

such

as

ThPP

or

glycerol

in

the

dialysis

medium

and

the

omission

of

selected

steps

from

the

procedure

Notably

better

reproducibility

in

rates

of

NADH

oxidation

were

obtained

using

mM

HEPES

buffer

-LRB-

pH

-RRB-

with

mM

DTT

Consequently

this

method

of

SEP

preparation

was

used

for

all

reported

results

The

low

rates

obtained

with

hexose

and

the

octulose

phosphates

were

initially

thought

to

be

due

to

depressed

activity

of

a

key

group-transferring

enzyme

particularly

transketolase

This

was

shown

to

be

unlikely

since

the

maximum

catalytic

activity

of

TK

as

well

as

that

of

several

other

enzymes

of

the

reductive

PP

in

SEP

was

sufficient

to

support

rates

of

fixation

in

excess

of

μmol

h

mg

Chl

-LRB-

Table

-RRB-

The

enzyme

activities

were

measured

between

and

h

after

chloroplasts

were

disrupted

and

are

representative

of

their

activities

in

SEP

during

progressive

or

serial

assays

of

NADH

oxidation.Table

catalytic

activity

of

selected

enzymes

of

the

reductive

PP

in

Spinach

SEPEnzymeCatalytic

activity

-LRB-

μmol

h

mg

Chl

-RRB-

This

studyData

of

Latzko

and

Gibbs

-LRB-

-RRB-

for

Spinach

leaf

extractPhosphoglycerate

dehydrogenase

-LRB-

NADP

-RRB-

l

m

Phosphotransferaseg

in

Pea

leaf

nPhosphotransferaseg

in

Chlorella

fusca

nPhosphotransferaseg

in

Spinach

leaf

extractNDn

mEnzymes

were

assayed

using

the

methods

of

a

b

Latzko

and

Gibbs

-LRB-

-RRB-

c

Brin

-LRB-

-RRB-

d

Bergmeyer

and

Bernt

-LRB-

-RRB-

e

Latzko

and

Gibbs

-LRB-

-RRB-

f

Woodrow

and

Walker

-LRB-

-RRB-

g

Arora

et

al.

-LRB-

-RRB-

h

Brand

-LRB-

-RRB-

i

Assayed

using

Ery

and

Xlu

as

substratesj

Assayed

using

Rib

and

Xlu

as

substratek

Assayed

using

the

Pi

liberation

methodl

Assayed

using

-LSB-

-RSB-

d-g-d-i-oct

and

Seh

as

substratesm

Assayed

using

-LSB-

-RSB-

d-g-d-a-oct

and

Seh

as

substratesn

Assayed

using

d-g-d-i-oct

and

Seh

as

substratesND

not

detected

Both

the

mono

and

bisphosphates

of

d-g-d-a-oct

were

marginally

more

active

substrates

for

the

support

of

fixation

-LRB-

Table

-RRB-

than

the

ido-oct

phosphate

esters

d-g-d-i-oct

was

least

effective

and

has

not

been

assigned

a

substrate

role

in

a

new

reaction

sequence

for

PS

presented

as

Scheme

and

is

a

conclusion

of

this

paper

This

is

a

curious

finality

to

this

study

of

the

octuloses

in

PS

since

d-g-d-i-oct

because

of

its

identification

in

the

L-type

PP

was

the

initial

substrate

of

choice

to

test

the

hypothesis

of

this

study

-LRB-

see

Introduction

-RRB-

Although

d-g-d-i-oct

has

a

degree

of

isotopic

equilibrium

with

all

the

octulose

phosphates

listed

in

Table

we

find

no

other

evidence

or

reason

to

now

support

its

inclusion

in

the

new

PS

scheme

d-g-d-a-oct

is

the

most

reactive

octulose

substrate

in

both

reductive

fixation

and

in

its

formation

from

the

monophosphate

by

PT

catalysis

in

the

reductive

direction

of

PS

-LRB-

see

later

discussion

on

PT

-RRB-

However

d-g-d-a-oct

forms

Rib

if

it

is

cleaved

by

the

ALD

activity

of

SEP

so

the

higher

fixation

by

d-g-d-a-oct

-LRB-

Table

-RRB-

may

in

part

be

attributed

to

some

Rib

release

d-g-d-i-oct

is

also

more

effective

in

the

support

of

fixation

-LRB-

Table

-RRB-

than

its

bisphosphate

and

it

is

also

independent

in

the

new

PS

scheme

of

any

need

to

transact

the

PT

step

at

reaction

XII

of

the

new

scheme

-LRB-

see

later

section

-RRB-

It

is

also

noted

that

the

Furbank

and

Lilley

-LRB-

-RRB-

assay

for

fixation

identified

slow

reacting

intermediates

in

the

reduction

assay

-LRB-

Table

-RRB-

These

intermediates

may

accumulate

and

thus

be

confined

to

a

linear

non-autocatalytic

-LRB-

Walker

and

Lilley

-RRB-

expression

of

PS

in

vitro

with

resultant

slow

synthesis

of

Ru

Determination

of

the

activity

of

the

non-oxidative

segment

of

the

PP

The

capacity

to

convert

Rib

to

hexose

is

the

measure

of

the

activity

of

the

enzymes

comprising

the

non-oxidative

segment

of

the

PP

-LRB-

Williams

et

al.

Fig.

-RRB-

When

the

products

of

this

conversion

are

removed

by

NADP

reduction

using

an

excess

of

PGI

and

Glc

the

reduction

rate

of

the

pyridine

nucleotide

is

the

measure

of

the

maximum

catalytic

capacity

of

the

preparation

-LRB-

see

Methods

-RRB-

The

average

max

rate

of

Rib

to

hexose

conversion

by

SEP

was

μmol

h

mg

Chl

-LRB-

S.D.

n

-RRB-

The

ability

of

SEP

from

spinach

chloroplasts

-LRB-

Furbank

and

Lilley

-RRB-

to

catalyse

the

above

process

-LRB-

half-life

h

-RRB-

was

greater

than

its

stability

in

measurements

of

the

reductive

PP

-LRB-

half-life

h

-RRB-

When

SEP

was

prepared

by

the

method

of

Kaiser

and

Bassham

-LRB-

-RRB-

it

catalysed

the

non-oxidative

segment

reactions

of

the

PP

at

the

rate

of

μmol

h

mg

Chl

and

this

was

enhanced

at

all

test

times

by

over

a

stability

time

course

by

the

inclusion

of

ThPP

-LRB-

mM

-RRB-

This

suggests

that

TK

may

have

been

susceptible

to

inactivation

by

the

assay

system

-LRB-

Murphy

and

Walker

-RRB-

However

comparison

of

these

data

with

the

above

results

for

the

reductive

PP

show

that

the

maximum

catalytic

capacity

of

the

non-oxidative

segment

of

PP

in

SEP

using

Rib

as

substrate

is

only

of

Rib

carboxylating

activity

in

the

reductive

PP

-LRB-

Table

-RRB-

The

above

finding

thus

discounts

the

possibility

that

the

complete

network

of

the

PP

-LRB-

Fig.

-RRB-

in

SEP

can

make

any

significant

overlapping

contribution

to

the

flux

of

the

reductive

PP

of

PS

Phosphotransferase

activity

The

proposed

alternative

mechanism

for

the

regenerative

phase

of

the

path

of

carbon

in

PS

-LRB-

Fig.

-RRB-

presents

a

number

of

features

in

common

with

the

L-type

PP

in

animal

tissues

-LRB-

Fig.

-RRB-

-LRB-

Williams

et

al.

Williams

-RRB-

In

particular

both

pathways

require

the

participation

of

a

specific

phosphotransferase

PT

-LRB-

see

Methods

-RRB-

The

presence

and

an

excess

of

PT

activity

to

support

the

maximum

flux

of

Rib

conversion

to

hexose

and

triose-P

in

vitro

was

reported

for

a

number

of

animal

and

plant

tissues

-LRB-

Arora

et

al.

-RRB-

PT

catalysed

the

reaction

of

Eq

-LRB-

-RRB-

using

the

same

rat

liver

enzyme

catalyst

that

generated

results

used

for

the

initial

hypothesis

of

the

classical

-LRB-

F-type

-RRB-

PP

-LRB-

Horecker

et

al.

Horecker

Williams

et

al.

-RRB-

PT

was

also

detected

in

French

Press

extracts

of

green

algae

-LRB-

Chlorella

fusca

-RRB-

and

enzyme

extracts

of

pea

leaf

-LRB-

Arora

et

al.

-RRB-

-LRB-

see

Table

-RRB-

The

PT

enzyme

has

not

previously

been

investigated

for

any

putative

role

in

the

metabolism

of

plants

The

detection

of

and

d-g-d-i-oct

in

spinach

leaves

-LRB-

Bartlett

et

al.

-RRB-

and

isolated

chloroplasts

following

short

periods

of

assimilation

-LRB-

this

paper

-RRB-

raised

the

question

of

the

origin

of

this

bisphosphate

compound

The

reaction

of

Eq

-LRB-

-RRB-

shows

the

formation

of

d-g-d-i-oct

and

the

data

reported

here

examine

the

activity

and

mechanism

of

phosphate

donor

and

transfer

for

the

formation

of

d-g-d-i-oct

In

particular

the

phosphotransferase

proposed

by

Williams

and

Clark

-LRB-

-RRB-

Clark

et

al.

-LRB-

-RRB-

and

Arora

et

al.

-LRB-

-RRB-

is

featured

The

L-type

PP

-LRB-

Fig.

-RRB-

and

the

hypothesized

path

of

carbon

in

PS

-LRB-

Fig.

-RRB-

proposed

a

phosphotransferase

which

catalysed

a

reversible

phosphate

transfer

between

octulose

and

sedoheptulose

phosphates

-LRB-

Eq

-LRB-

-RRB-

-RRB-

The

investigations

reported

on

this

reaction

were

made

using

spinach

leaf

extracts

and

SEP

preparations

from

spinach

chloroplasts

PT

activity

was

not

detected

using

either

spinach

leaf

extract

or

SEP

to

catalyse

the

reaction

of

Eq

-LRB-

-RRB-

when

the

measurements

were

performed

in

the

oxidative

direction

of

d-g-d-i-oct

formation

-LRB-

Fig.

-RRB-

A

study

of

SEP-catalysed

phosphate

transfer

from

Seh

to

-LSB-

-RSB-

d-g-d-i-oct

-LRB-

Eq

-LRB-

-RRB-

in

the

reductive

direction

of

the

PS

pathway

-RRB-

using

the

radiochromatographic

stop

assay

procedure

-LRB-

see

Methods

-RRB-

showed

PT

activity

was

μmol

h

mg

Chl

-LRB-

S.D.

n

-RRB-

-LRB-

Table

-RRB-

This

value

exceeded

the

maximum

rate

of

SEP-catalysed

conversion

of

Rib

to

hexose

phosphates

but

was

only

of

the

chloroplast

fixation

rate

required

by

the

PT

reaction

step

-LRB-

See

New

Scheme

-RRB-

and

of

the

degree

of

carboxylation

required

for

the

ab

initio

synthesis

of

octulose

-LRB-

-RRB-

by

PS

d-g-d-a-oct

was

a

more

effective

substrate

in

this

PT

assay

with

activity

of

μmol

h

mg

Chl

-LRB-

P.E.

n

see

Table

-RRB-

which

is

of

the

required

chloroplast

fixation

rate

at

the

PT

step

of

the

revised

RPP

scheme

and

of

the

activity

necessary

for

octulose

formation

Significant

PT

activity

was

found

in

spinach

leaf

extract

-LRB-

μmol

h

mg

Chl

see

Table

-RRB-

using

the

above

radiochromatographic

method

and

the

-LSB-

-RSB-

d-g-d-a-oct

substrate

-LRB-

Eq

-LRB-

-RRB-

-RRB-

No

activity

was

detected

in

the

leaf

extract

using

d-g-d-i-oct

and

oxidative

direction

assays

-LRB-

see

Table

-RRB-

However

it

is

not

concluded

that

positive

PT

measurement

in

the

reductive

PS

mode

is

evidence

for

an

exclusive

unidirectional

flux

mechanism

Rather

the

failure

in

the

application

of

the

oxidative-mode

methods

-LRB-

Arora

et

al.

-RRB-

is

attributed

inter

alia

to

subversively

high

levels

of

Mg

and

DTT

in

SEP.

A

test

of

the

absolute

specificity

of

the

PT

reaction

was

made

using

-LSB-

-RSB-

Seh

substrate

and

d-g-d-i-oct

The

reaction

was

carried

out

using

SEP

and

exactly

the

same

conditions

as

the

above

radiochromatographic

stop

assay

All

products

of

the

reaction

were

resolved

using

formate

ion-exchange

chromatography

-LRB-

see

Methods

-RRB-

and

their

radioactivity

measured

which

coincided

with

the

elution

peak

of

the

authentic

compound

was

recovered

and

its

identity

further

confirmed

by

rechromatography

with

DEAE-Sephadex

-LRB-

borate

form

-RRB-

-LRB-

Kapuscinski

et

al.

-RRB-

Aldolase

cleavage

of

the

purified

Oct

and

isolation

of

using

short

column

-LRB-

-RRB-

formate

ion-exchange

chromatography

showed

that

of

the

incorporated

was

in

this

compound

The

results

of

this

experiment

indicated

that

qualitatively

SEP

has

enzymatic

activity

capable

of

specifically

phosphorylating

d-g-d-i-oct

at

position

using

Seh

as

the

phosphate

donor

There

was

also

an

extensive

conversion

of

the

-LSB-

-RSB-

Seh

to

and

formation

of

-LSB-

-RSB-

Fru

Only

of

the

-LSB-

-RSB-

Seh

substrate

remained

after

min

of

reaction

time

When

mM

and

mM

DTT

were

included

in

the

above

reaction

mixture

of

the

label

of

-LSB-

-RSB-

Seh

was

converted

to

and

only

to

the

isotopically

labelled

d-g-d-i-oct

It

is

suggested

that

high

concentrations

of

in

SEP

may

have

activated

chloroplastic

bisphosphatases

and

account

in

part

or

completely

for

an

inability

to

demonstrate

significantly

higher

activities

of

PT

Inclusion

of

DTT

and

the

endogenous

levels

of

aldolase

in

SEP

and

spinach

leaf

extracts

possibly

acted

to

further

diminish

the

full

expression

of

the

activity

of

the

PT

enzyme

Hence

the

minimum

qualification

for

the

measurements

of

the

crude

activity

of

PT

During

the

conduct

of

the

above

investigations

and

measurements

of

PT

activity

the

various

SEP

preparations

were

also

used

to

test

the

possibility

that

other

intermediary

compounds

and

enzymes

may

act

to

phosphorylate

d-g-d-i-oct

Among

the

test

substrates

were

-LSB-

-RSB-

ATP

inorganic

pyrophosphate

Fru

and

Ru

The

following

purified

enzymes

and

enzyme

preparations

were

also

investigated

PFK

from

rabbit

muscle

phosphoribulokinase

-LRB-

EC

-RRB-

from

spinach

the

endogenous

kinases

of

SEP

using

-LSB-

-RSB-

ATP

as

substrate

and

sedoheptulose

In

summary

of

these

enzymes

or

substrates

was

effective

except

for

muscle

PFK

which

catalysed

the

formation

of

-LSB-

-RSB-

d-g-d-i-oct

from

the

reaction

of

-LSB-

-RSB-

ATP

and

d-g-d-i-oct

Notwithstanding

this

when

SEP

was

used

to

catalyse

the

same

reaction

only

a

miniscule

rate

of

labelled

d-g-d-i-oct

formation

was

measured

-LRB-

μmol

h

mg

Chl

-RRB-

PFK

accepts

Fru

and

Seh

as

substrates

-LRB-

Karadsheh

et

al.

-RRB-

and

on

the

basis

of

the

above

finding

d-g-d-i-oct

may

be

added

to

the

list

However

the

maximum

catalytic

activity

of

PFK

in

spinach

chloroplasts

is

only

μmol

h

mg

Chl

-LRB-

Kelly

and

Latzko

-RRB-

and

it

is

therefore

unlikely

to

be

a

significant

contributor

to

d-g-d-i-oct

formation

in

RPP

reactions

in

PS

The

results

of

this

section

-LRB-

Table

-RRB-

showed

that

minimum

estimates

of

PT

were

compromised

when

SEP

is

the

source

of

activity

The

results

also

showed

that

d-g-d-a-oct

phosphate

was

the

most

active

substrate

and

that

PT

may

be

the

rate-limiting

enzyme

for

octulose

involvement

in

path

of

carbon

reactions

This

limitation

in

minimum

estimates

of

PT

activity

in

SEP

-LRB-

Table

-RRB-

may

account

for

altro-octuloses

being

restricted

to

the

support

of

only

of

the

rate

of

fixation

by

intact

spinach

chloroplasts

and/or

of

the

maximum

rate

required

at

the

PT

reaction

step

-LRB-

See

later

discussion

of

the

new

reactions

in

Scheme

-RRB-

Arabinose

reactions

with

spinach

stromal

enzyme

preparation

In

the

L-type

PP

-LRB-

Fig.

-RRB-

the

prior

formation

of

Ara

and

its

use

by

aldolase

is

the

key

step

for

the

formation

of

d-g-d-i-oct

In

contrast

the

hypothetical

mode

of

d-g-d-i-oct

synthesis

and

reaction

in

the

PS

scheme

of

Fig.

results

in

the

later

formation

of

Ara

and

its

contribution

to

Rib

-LRB-

via

Ru

-RRB-

Thus

it

was

anticipated

that

Ara

by

its

initial

assignment

of

an

intermediary

role

in

the

flux

of

the

pathway

of

Fig.

should

support

fixation

in

the

SEP

assay

system

The

result

-LRB-

Table

-RRB-

of

the

test

of

this

proposition

was

negative

Arabinose

as

a

single

test

substrate

had

negligible

ability

to

support

fixation

in

SEP-catalysed

reductive

PP

reactions

in

PS

A

second

question

raised

by

the

hypothetical

scheme

of

Fig.

is

whether

SEP

is

able

to

catalyse

Ara

dissimilation

and

interconvert

it

into

other

intermediates

of

either

the

Calvin

Pathway

or

octulose

phosphates

SEP

catalysed

the

incorporation

of

radioactivity

from

-LSB-

-RSB-

Ara

into

the

carbons

of

the

following

sugar

phosphate

intermediates

of

the

reductive

PP

triose-P

ketopentose

hexose

and

heptulose

mono

and

bisphosphates

-LRB-

Fig.

-RRB-

Fig.

showing

the

spinach

SEP-catalysed

conversion

of

-LSB-

-RSB-

Ara

in

the

presence

of

a

nine-fold

excess

of

unlabelled

Rib

to

other

radioactively

labelled

sugar

phosphates

-LSB-

-RSB-

Ara

and

Rib

were

incubated

for

h

with

spinach

SEP

as

detailed

in

Materials

and

methods

The

products

of

the

reaction

were

separated

by

descending

paper

chromatography

using

the

solvent

Lanes

and

of

the

chromatogram

show

the

mixture

of

sugar

phosphates

formed

from

Ara

dissimilation

Lane

shows

the

radioautograph

of

products

in

the

absence

of

Rib

Lane

shows

an

absence

of

product

formation

when

boiled

SEP

was

used

The

identity

by

visualization

of

the

sugar

phosphates

at

Lanes

and

was

established

by

comparison

with

the

positions

-LRB-

not

shown

in

Fig.

-RRB-

of

the

following

sugar

phosphate

marker

standards

that

accompanied

the

four

lanes

of

the

chromatogram

Fru

-LRB-

FBP

-RRB-

Seh

Glc

Fru

Ara

DHAP

Ru

Xlu

The

one-dimensional

chromatography

system

used

does

not

permit

the

identification

of

octulose

phosphates

-LRB-

see

Kapuscinski

et

al.

for

a

list

of

Rp-i

values

for

some

of

the

above

sugar

phosphates

resolved

by

the

solvent

-RRB-

The

visualization

and

tentative

identification

of

the

labelled

products

was

distinct

except

for

the

band

running

just

ahead

of

Ara

which

may

have

involved

ketopentose

and

DHAP

However

into

these

products

only

occurred

in

the

presence

of

an

excess

-LRB-

nine-fold

-RRB-

of

unlabelled

Rib

The

process

only

faintly

labelled

Rib

When

mM

-LSB-

-RSB-

Ara

was

the

sole

substrate

its

conversion

to

other

identified

sugar

phosphates

was

only

and

there

was

clear

incorporation

into

an

unresolved

band

of

phosphorylated

compounds

with

an

Rf

value

coincident

with

the

ketopentose

phosphates

and

also

close

to

DHAP

-LRB-

Fig.

-RRB-

It

was

imperative

to

ensure

that

into

the

sugar

phosphates

was

not

the

result

of

non-enzymatic

conversions

occurring

during

sample

processing

This

possibility

was

eliminated

on

the

basis

of

several

control

experiments

which

included

different

ways

of

terminating

the

reaction

-LRB-

boiling

and

perchloric

acid

treatment

-RRB-

and

incubations

with

SEP

boiled

before

the

addition

of

-LSB-

-RSB-

Ara

When

the

incubations

were

carried

out

with

SEP

at

pH

in

the

presence

of

mM

and

mM

DTT

-LRB-

conditions

favouring

activation

of

some

key

regulatory

enzymes

of

the

reductive

PP

-RRB-

there

was

no

incorporation

of

isotope

from

-LSB-

-RSB-

Ara

-LRB-

and

excess

Rib

-RRB-

into

any

other

metabolites

Since

the

experiments

used

SEP

that

had

provided

approx

incorporation

from

-LSB-

-RSB-

Ara

-LRB-

in

the

presence

of

a

nine-fold

excess

of

Rib

-RRB-

into

hexose

and

by

visual

judgement

of

the

chromatogram

incorporations

into

Triose-P

Ru

Fru

and

Seh-bisphosphates

it

was

concluded

that

or

the

reductant

may

be

inhibitory

These

latter

observations

were

not

pursued

further

The

above

is

the

first

report

of

Ara

reaction

in

a

chloroplast

preparation

Arabinose

is

a

competitive

inhibitor

of

TA-catalysed

reactions

with

a

Ki

of

μM

-LRB-

Williams

et

al.

-RRB-

d-manno-Heptulose

a

product

of

TK

catalysis

using

a

ketosugar-P

donor

and

Ara

is

a

competitive

inhibitor

of

TK-catalysed

reactions

involving

Ery

-LRB-

Arora

-RRB-

Moreover

in

all

animal

and

neoplastic

tissues

investigated

-LRB-

Williams

et

al.

-RRB-

Ara

acted

as

a

powerful

competitive

inhibitor

-LRB-

Ki

μM

Arora

-RRB-

when

the

tissue

enzyme

preparations

converted

Rib

to

hexose

and

triose

phosphates

using

standard

assay

conditions

for

the

non-oxidative

PP

-LRB-

Methods

section

-RRB-

Thus

it

was

reasoned

that

enzyme

inhibition

may

be

responsible

for

the

failure

to

find

uncontentious

evidence

that

Ara

is

a

utilized

substrate

in

reactions

catalysed

by

spinach

SEP.

This

proposition

was

tested

with

SEP

using

-LSB-

Ara

-RSB-

-LSB-

Rib

-RSB-

ratios

ranging

from

to

Some

inhibition

was

found

but

it

was

notably

less

than

that

uniformly

encountered

with

animal

and

neoplastic

tissue

enzyme

preparations

Even

a

mixture

of

Ara

and

Rib

with

SEP

exhibited

of

the

control

rate

measured

with

Rib

alone

This

small

degree

of

inhibition

is

therefore

not

an

explanation

for

the

inability

of

SEP

to

catalyse

Ara

as

a

substrate

in

carboxylation

reactions

In

experiments

that

involved

incubation

of

SEP

with

unlabelled

Ara

and

slow

fixation

occurred

-LRB-

μmol

h

mg

Chl

-RRB-

Replacement

of

Ara

with

Rib

resulted

in

a

rapid

rate

of

formation

of

acid

stable

product

consistent

with

the

peak

activity

of

Rib

in

reductive

PP

reactions

as

shown

by

the

data

of

Table

Moreover

using

the

polarographic

method

for

measuring

evolution

-LRB-

Methods

section

-RRB-

and

intact

spinach

chloroplasts

it

was

found

that

the

inclusion

of

mM

Rib

stimulated

the

rate

of

evolution

by

and

the

same

concentration

of

Ara

stimulated

the

rate

by

Schafer

et

al.

-LRB-

-RRB-

found

that

free

Ara

and

Rib

were

transported

into

intact

chloroplasts

at

equal

rates

and

the

above

data

suggest

that

spinach

chloroplasts

may

be

differentially

permeable

to

the

phosphates

with

Rib

being

times

more

accessible

and

effective

than

Ara

There

is

also

controversy

about

the

substrate

role

of

d-Ara

in

higher

organisms

-LRB-

a

brief

statement

of

its

intermediary

metabolic

history

is

given

in

Williams

et

al.

-RRB-

Interconversion

of

Ara

with

other

pentose

phosphates

-LRB-

via

isomerization

to

Ru

-RRB-

by

microorganisms

has

been

known

for

a

considerable

time

-LRB-

Volk

-RRB-

but

the

direct

evidence

for

such

a

process

in

rat

liver

was

first

demonstrated

by

Bleakley

et

al.

-LRB-

-RRB-

The

enzyme

equilibrating

Ara

with

other

pentose

phosphates

was

originally

proposed

to

be

of

the

epimerase

type

-LRB-

Williams

and

Clark

-RRB-

however

work

with

pig

liver

suggested

that

utilization

and

conversion

of

Ara

to

other

metabolites

may

proceed

via

Ru

-LRB-

Williams

unpublished

results

-RRB-

The

essence

of

the

controversy

concerning

the

metabolism

of

Ara

is

not

so

much

its

formation

and

presence

in

plant

and

other

tissues

but

its

comparatively

low

rate

of

substrate

utilization

by

tissue

enzyme

preparations

in

vitro

usually

only

at

of

the

rate

of

Rib

dissimilation

This

is

the

case

reported

here

where

SEP

catalysed

utilization

of

Ara

was

extremely

low

This

controversy

was

settled

when

Bartlett

et

al.

-LRB-

-RRB-

and

Flanagan

et

al.

-LRB-

-RRB-

demonstrated

the

role

that

catalytic

quantities

of

Ara

and

Rib

were

able

to

play

in

TA

and

ALD

exchange

reactions

These

exchange

reactions

affected

epimerase

interconversions

of

d-g-d-i-oct

and

d-g-d-a-oct

and

their

bisphosphates

It

was

shown

that

Ara

was

reversibly

exchanged

into

positions

of

the

octulose

phosphates

with

exchange

rates

that

were

greater

than

the

mass

transfer

flux

rates

catalysed

by

the

maximum

catalytic

capacities

of

each

of

the

group

transferring

enzymes

TA

and

ALD

acting

in

the

reactions

of

the

following

equations

The

roles

of

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

are

shown

at

reactions

IX

and

X

in

the

following

new

reaction

sequence

-LRB-

Scheme

-RRB-

for

the

path

of

carbon

in

PS

Taken

together

these

reactions

catalyse

the

epimerization

of

the

ido

and

altro-octulose

monophosphates

Equation

-LRB-

-RRB-

shows

the

interconversion

of

ido

and

altro-octulose

bisphosphates

by

the

aldolase

exchange

reaction

-LRB-

Bartlett

et

al.

Flanagan

et

al.

-RRB-

It

is

proposed

that

the

reaction

of

assists

in

the

prevention

of

an

accumulation

of

the

much

slower

reacting

d-g-d-i-oct

by

rapidly

channelling

its

carbon

into

the

more

reactive

epimeric

form

d-g-d-a-oct

-LRB-

see

XII

and

XIII

in

Scheme

I

involving

octulose

phosphates

in

PS

-RRB-

The

experimental

results

of

this

paper

and

the

reactions

of

IX

XII

and

XIII

in

the

reaction

scheme

suggest

that

d-g-d-a-oct

mono

and

bisphosphates

become

the

reactive

forms

of

ido-octulose

phosphates

by

the

reactions

of

-LRB-

-RRB-

and

-LRB-

-RRB-

using

Rib

and

Ara

as

cycling

catalytic

co-factors

in

reactions

catalysed

by

TAx

and

Aldx

We

now

discount

any

role

for

d-glycero-d-ido-octulose

as

was

initially

proposed

for

the

Fig.

hypothesis

The

revised

scheme

only

differs

significantly

from

Fig.

at

reaction

IX

where

epimerization

of

d-g-d-i-oct

to

d-g-d-a-oct

occurs

From

that

point

ido-octulose

phosphates

are

omitted

from

any

further

involvement

in

the

completion

of

the

cycle

Using

PT

activity

data

with

d-g-d-a-oct

as

substrate

-LRB-

Table

-RRB-

it

is

calculated

that

the

revised

sequence

may

make

a

contribution

that

ranges

from

-LRB-

-RRB-

based

on

the

rate

of

fixation

-LRB-

μmol

h

mg

Chl

-RRB-

in

intact

chloroplasts

to

a

maximum

of

-LRB-

-RRB-

based

on

the

required

fixation

rate

-LRB-

μmol

h

mg

Chl

-RRB-

at

the

PT

step

-LRB-

reaction

XIII

-RRB-

of

Scheme

for

the

RPP

Scheme

Finally

it

is

concluded

that

metabolic

function

has

directed

a

distinct

and

confining

intermediary

metabolism

on

ido

and

altro-octulose

phosphates

In

the

L-type

PP

-LRB-

Fig.

-RRB-

selection

of

d-glycero-d-ido-octulose

phosphates

realised

the

formation

of

the

metabolically

active

products

Fru

and

Glc

d-glycero-d-altro-Octulose

phosphates

in

the

pathway

of

Fig.

will

only

lead

to

the

formation

of

Fru

and

the

dead-end

product

d-allose

In

Scheme

formulated

here

the

formation

of

d-g-d-a-oct

leads

to

the

production

of

Rib

the

most

active

intermediate

for

the

support

of

fixation

-LRB-

Table

-RRB-

Formation

and

further

utilization

of

d-g-d-i-oct

by

the

reaction

scheme

would

produce

stoichiometric

amounts

of

Ara

which

as

a

metabolic

product

has

no

ability

to

fix

-LRB-

-RRB-

calvin cycle
chloroplasts
spinach leaf
octulose phosphates
phosphotransferase

arabinose 5- phosphate
14c-carbon dioxide
14c-labelled novel compounds
keto-group exchange
l-type pentose pathway
octulose-p epimerization
revised rpp in ps
stromal enzymes