A

re-investigation

of

the

path

of

carbon

in

photosynthesis

utilizing

GC/MS

methodology

Unequivocal

verification

of

the

participation

of

octulose

phosphates

in

the

pathway

A

GC/EIMS/SIM

methodology

has

been

developed

to

re-examine

the

path

of

carbon

in

photosynthesis

Exposing

isolated

spinach

chloroplasts

to

on

a

solid

support

for

a

defined

period

followed

by

quenching

and

work-up

provided

a

mixture

of

labelled

sugar

phosphates

After

enzymatic

dephosphorylation

and

derivatization

the

Mox-TMS

sugars

were

analysed

using

the

above

method

The

purpose

of

the

study

was

to

try

to

calculate

the

atom

enrichment

of

in

as

many

of

the

individual

carbons

in

each

of

the

derivatized

sugars

as

was

practical

using

diagnostic

fragment

ions

In

the

event

only

one

s

experiment

provided

sufficient

data

to

enable

a

range

of

enrichment

values

to

be

calculated

This

confirmed

that

D-glycero-D-altro-octulose

phosphate

was

present

in

the

chloroplasts

and

was

heavily

labelled

in

the

and

positions

in

keeping

with

the

hypothesis

that

it

had

an

inclusive

role

and

a

labelling

pattern

consistent

with

a

new

modified

pathway

of

carbon

in

photosynthesis

Introduction

In

two

previous

papers

we

described

a

GC/MS

methodology

which

is

capable

of

providing

a

rapid

specific

and

quantitative

means

of

analysis

of

incorporation

from

into

those

sugar

phosphates

involved

in

the

path

of

carbon

in

photosynthesis

-LRB-

Irvine

et

al.

MacLeod

et

al.

-RRB-

This

provided

a

framework

for

a

re-investigation

of

the

photosynthetic

carbon

reduction

-LRB-

PCR

-RRB-

pathway

in

plants

commonly

known

as

the

Calvin

Cycle

-LRB-

Calvin

-RRB-

Although

the

PCR

pathway

as

presented

by

Calvin

-LRB-

Fig.

-RRB-

is

widely

accepted

its

re-examination

is

warranted

for

the

following

reasons

-LRB-

i

-RRB-

the

evidence

upon

which

Calvin

based

the

pathway

was

incomplete

-LRB-

ii

-RRB-

more

recent

investigations

support

the

inclusion

of

other

sugar

phosphates

notably

octulose

phosphates

in

the

pathway

-LRB-

Williams

and

MacLeod

preceding

paper

-RRB-

and

-LRB-

iii

-RRB-

the

substantial

advances

in

analytical

technologies

since

Calvin

's

data

was

collected

in

the

using

and

paper

chromatography

have

the

potential

to

provide

more

definitive

and

comprehensive

results

than

were

possible

for

Calvin

to

obtain

Fig.

path

of

carbon

in

photosynthesis

-LRB-

Calvin

-RRB-

Each

asterisk

-LRB-

-RRB-

represents

a

carbon

atom

labelled

during

one

turn

of

the

cycle

For

explanation

of

abbreviations

see

preceding

paper

This

paper

describes

the

measurement

using

GC/EIMS/SIM

of

incorporation

into

individual

carbon

atoms

of

to

sugar

phosphates

following

short-term

exposure

of

isolated

chloroplasts

to

during

PS

Materials

and

methods

Chemicals

Sorbitol

was

purchased

from

Koch-Light

UK

sodium

pyrophosphate

from

Mallinckrodt

USA

and

Percoll

from

Pharmacia

Sweden

Enzymes

and

cofactors

were

obtained

from

either

Sigma

USA

or

Boehringer-Mannheim

Germany

Ion

exchange

resins

were

obtained

from

BioRad

USA

Analytical

grade

chemicals

were

obtained

from

Ajax

Australia

BDH

UK

or

Merck

Germany

All

solvents

were

distilled

before

use

while

pyridine

was

pre-dried

and

distilled

from

calcium

hydride

under

argon

Water

was

obtained

from

a

Millipore

Milli-Q

system

The

gas

-LRB-

atom

enriched

-RRB-

was

purchased

from

Cambridge

Isotopes

UK

and

prepared

to

in

nitrogen

in

a

gas

cylinder

A

second

cylinder

was

made

up

with

unenriched

in

nitrogen

Gas

analysis

of

the

cylinders

was

carried

out

on

a

Varian

gas

chromatograph

using

a

m

cm

glass

column

packed

with

Porapak

N

mesh

stationary

phase

Plant

material

Spinach

-LRB-

Spinacia

oleracea

Yates

Hybrid

-RRB-

seeds

were

obtained

from

Henderson

Seed

Co.

Pty.

Ltd.

Lower

Templestowe

Vic.

Australia

Growth

of

plant

material

chloroplast

isolation

chlorophyll

assay

measurement

of

chloroplast

intactness

polarographic

measurement

of

chloroplast

activity

in

suspension

The

above

methods

have

been

described

in

detail

in

the

accompanying

paper

-LRB-

Williams

and

MacLeod

preceding

paper

-RRB-

Measurement

of

chloroplast

activity

on

filter

membranes

The

activity

of

the

isolated

chloroplasts

mounted

on

a

filter

membrane

was

measured

polarographically

using

a

Clark

oxygen

electrode

adapted

for

measurement

of

the

oxygen

evolution

from

leaf-discs

-LRB-

Delieu

and

Walker

-RRB-

Methods

for

the

preparation

of

chloroplasts

for

use

in

the

leaf-disc

oxygen

electrode

were

based

on

those

previously

described

-LRB-

Cerovic

et

al.

-RRB-

Chloroplasts

were

mounted

on

a

filter

membrane

by

filtering

a

suspension

of

chloroplasts

through

a

cellulose

nitrate

membrane

-LRB-

Sartorius

mm

diameter

pore

size

μm

-RRB-

This

was

accomplished

using

a

Millipore

solvent

clarification

apparatus

fitted

with

a

ml

funnel

A

suspension

of

chloroplasts

equivalent

to

μg

chlorophyll

in

ml

resuspension

medium

at

pH

containing

mM

sorbitol

mM

HEPES-KOH

mM

EDTA

mM

and

mM

was

allowed

to

filter

through

the

membrane

under

gravity

or

with

a

very

light

suction

so

that

the

chloroplasts

became

trapped

on

the

surface

of

the

membrane

in

a

thin

film

of

resuspension

medium

The

chloroplasts

were

washed

with

a

further

ml

of

the

resuspension

medium

and

then

given

a

final

wash

with

ml

of

assay

medium

containing

mM

inorganic

phosphate

U

ml

alkaline

phosphatase

-LRB-

Sigma

human

placenta

-RRB-

and

U

ml

catalase

in

resuspension

medium

A

disc

of

-LRB-

representing

of

the

chloroplasts

-RRB-

was

cut

from

the

membrane

and

mounted

in

a

leaf-disc

oxygen

electrode

-LRB-

Hansatech

-RRB-

Once

assembled

the

leaf-disc

oxygen

electrode

was

charged

with

an

atmosphere

of

in

at

atmospheric

pressure

and

sealed

The

chloroplasts

were

exposed

to

incandescent

light

from

a

pair

of

bifurcated

fibre

optic

light

sources

at

an

intensity

of

μEm

s

measured

at

the

underside

of

the

leaf-disc

electrode

light

window

The

temperature

of

the

water

bath

supplying

the

water

jacket

which

regulated

the

oxygen

electrode

temperature

was

maintained

at

°C

After

a

lag

period

of

approximately

min

the

evolution

of

could

be

monitored

as

a

digital

voltage

on

the

oxygen

electrode

control

box

and

was

also

recorded

on

a

Goerz

Metrawatt

SE

chart

recorder

The

rate

of

evolution

reached

a

linear

and

maximum

rate

at

approximately

min

after

switching

on

the

lights

The

rate

was

calculated

from

the

linear

portion

of

the

trace

Experiments

in

the

leaf-disc

oxygen

electrode

Chloroplasts

were

prepared

for

experiments

in

the

leaf

disc

oxygen

electrode

-LRB-

as

discussed

for

the

assay

of

chloroplast

activity

on

filter

membranes

in

that

equipment

-RRB-

and

a

linear

rate

of

evolution

was

established

At

this

time

a

volume

-LRB-

ml

-RRB-

of

in

was

flushed

through

the

ml

chamber

of

the

oxygen

electrode

that

contained

the

membrane-mounted

chloroplasts

and

after

resealing

the

chamber

the

chloroplasts

were

allowed

to

photosynthesize

in

the

atmosphere

for

a

prescribed

period

of

time

before

the

reactions

were

terminated

using

the

quenching

methods

described

below

Experiments

were

conducted

for

and

s

exposure

to

in

and

the

control

experiment

was

conducted

with

chloroplasts

exposed

to

in

for

s

in

the

dark

In

addition

a

blank

experiment

was

conducted

in

which

the

full

extraction

procedure

was

followed

in

the

absence

of

chloroplasts

to

measure

the

extent

of

any

contaminants

that

may

interfere

with

the

analysis

Five

repetitions

of

each

experiment

were

carried

out

using

the

same

chloroplast

preparation

Reactions

were

terminated

by

dissembling

the

apparatus

as

quickly

as

possible

and

plunging

the

chloroplast-containing

membrane

into

a

beaker

of

liquid

nitrogen

It

was

estimated

that

this

procedure

took

no

more

than

s

The

beaker

containing

the

membrane

was

then

stored

in

a

freezer

at

°C

where

the

liquid

nitrogen

was

allowed

to

evaporate

After

overnight

storage

at

°C

the

enzymes

were

denatured

and

the

metabolites

were

extracted

by

removing

the

beaker

containing

the

membranes

from

the

freezer

and

immediately

adding

boiling

ethanol

-LRB-

ml

-RRB-

and

boiling

for

a

further

min

The

membranes

were

then

removed

from

the

extract

and

thoroughly

rinsed

with

water

adding

the

washings

to

the

extract

After

cooling

the

pooled

extracts

were

then

evaporated

to

dryness

at

°C

under

a

stream

of

dry

nitrogen

Design

of

a

specific

photosynthesis

apparatus

Because

of

the

limitations

of

the

oxygen

electrode

for

the

type

of

experiments

that

were

required

it

was

decided

to

design

and

construct

an

apparatus

better

suited

for

the

purpose

The

basic

requirements

in

the

design

and

construction

of

the

photosynthetic

apparatus

were

-LRB-

-RRB-

that

the

chloroplasts

be

mounted

on

a

filter

membrane

inside

a

small

chamber

with

a

transparent

window

for

the

admission

of

light

-LRB-

-RRB-

the

filter

membrane

should

be

large

enough

to

hold

sufficient

chloroplasts

to

provide

enough

extracted

material

for

GC/MS

analysis

-LRB-

-RRB-

the

atmosphere

above

the

membrane

must

allow

the

establishment

of

steady

state

photosynthesis

with

unenriched

and

the

rapid

replacement

of

this

with

the

same

concentration

of

whilst

causing

minimum

disturbance

to

the

steady

state

-LRB-

-RRB-

the

volume

of

the

chamber

above

and

below

the

membrane

should

be

small

in

order

to

minimize

the

time

taken

to

flush

out

the

with

using

a

reasonable

gas

flow

rate

Also

the

volume

of

the

chamber

below

the

membrane

must

be

small

in

order

to

minimize

the

dead

volume

of

residual

and

-LRB-

-RRB-

there

must

be

provision

for

an

appliance

to

inject

a

liquid

to

quench

the

photosynthetic

reactions

after

a

given

time

and

for

the

convenient

removal

of

the

chloroplast

extract

after

the

quench

Filter

holders

were

available

-LRB-

Sartorius

SM

-RRB-

which

with

minimal

modification

provided

a

suitable

basis

on

which

to

construct

a

prototype

photosynthesis

apparatus

The

rapid

replacement

of

unenriched

with

was

provided

for

by

the

inclusion

of

two

gas

inlet

ports

each

supplying

an

internal

gas

manifold

around

the

outer

perimeter

of

the

chloroplast-supporting

membrane

The

manifolds

directed

across

the

entire

membrane

and

drove

excess

gas

out

through

a

series

of

gas

outlet

holes

drilled

in

the

housing

above

the

centre

of

the

membrane

which

vented

the

expelled

gases

to

the

atmosphere

An

electro-mechanical

timer

unit

was

constructed

to

permit

the

automatic

and

rapid

switching

of

the

gas

supply

from

a

gas

cylinder

containing

unenriched

in

nitrogen

to

another

cylinder

containing

in

nitrogen

via

a

pair

of

electrically

operated

gas

valves

causing

minimal

disruption

to

the

steady

state

of

photosynthesis

The

inlet

and

outlet

ports

for

the

filter

holder

provided

suitable

ports

for

the

entry

and

withdrawal

of

the

quenching

solution

A

ml

syringe

containing

the

quenching

solution

was

attached

to

the

filter

inlet

port

via

a

tap

which

could

be

opened

to

allow

the

injection

of

the

quenching

solution

at

the

completion

of

the

photosynthesis

experiment

By

applying

a

gentle

suction

via

a

tap

at

the

filter

outlet

port

the

extract

could

then

be

withdrawn

into

a

flask

containing

boiling

ethanol

located

below

the

apparatus

The

chloroplasts

and

apparatus

were

protected

from

the

heat

produced

by

the

hotplate

by

the

provision

of

a

reflective

heat

shield

Light

was

provided

via

fibre

optic

cables

which

greatly

reduced

the

transmission

of

heat

from

the

light

source

and

reduced

the

need

for

cooling

of

the

apparatus

The

concentrations

reported

for

chloroplast

metabolites

are

variable

but

have

usually

been

found

to

be

in

the

range

from

to

nmol

per

mg

chlorophyll

-LRB-

Gerhardt

et

al.

Giersch

et

al.

Giersch

-RRB-

A

filter

membrane

loaded

with

chloroplasts

equivalent

to

μg

of

chlorophyll

should

therefore

contain

nmol

of

most

metabolites

of

interest

Assuming

minimal

losses

during

processing

for

GC/MS

analysis

this

was

considered

to

be

adequate

chloroplast

material

for

each

experiment

Experiments

in

the

photosynthesis

apparatus

Chloroplasts

were

prepared

on

a

filter

membrane

as

described

for

the

experiments

in

the

oxygen

electrode

The

loaded

membrane

was

mounted

intact

in

the

photosynthesis

apparatus

A

gas

mixture

consisting

of

unenriched

in

nitrogen

was

applied

to

the

two

gas

inlet

ports

via

an

electrically

operated

valve

connected

to

a

specially

made

electro-mechanical

timer

unit

The

gas

stream

-LRB-

at

a

flow

rate

of

min

-RRB-

was

humidified

by

bubbling

it

through

a

tube

of

water

before

entering

the

valve

The

chloroplasts

were

illuminated

through

the

transparent

window

in

the

apparatus

by

a

pair

of

W

tungsten

lamps

each

directed

through

a

bifurcated

fibre

optic

light

guide

The

gas

mixture

was

supplied

for

min

before

the

timer

operated

the

valves

supplying

the

gas

and

switched

to

the

source

supplying

in

for

the

prescribed

period

Experiments

were

conducted

for

and

s

exposure

to

in

and

the

control

experiment

was

conducted

with

chloroplasts

exposed

to

in

in

the

dark

for

s

The

reactions

were

terminated

and

the

metabolites

were

extracted

by

injecting

hot

ethanol

-LRB-

ml

-RRB-

into

the

apparatus

and

allowing

the

chloroplasts

to

stand

in

the

solution

for

s

The

ethanolic

chloroplast

extract

was

then

drawn

through

the

membrane

using

gentle

suction

into

a

flask

that

contained

boiling

ethanol

-LRB-

ml

-RRB-

The

extraction

process

was

repeated

with

a

further

volume

of

boiling

ethanol

-LRB-

ml

-RRB-

followed

by

boiling

water

-LRB-

ml

-RRB-

The

combined

extract

was

then

boiled

for

min

and

allowed

to

cool

After

cooling

the

extract

was

evaporated

to

dryness

at

°C

using

a

stream

of

dry

gas

Preparation

of

samples

for

GC/MS

analysis

The

dried

samples

from

the

photosynthesis

experiments

described

above

were

redissolved

in

water

-LRB-

ml

-RRB-

and

then

processed

for

GC/MS

analysis

according

to

the

following

methods

The

chloroplast

extract

to

be

analyzed

by

GC/MS

was

applied

to

an

Elut

Bond

strong

anion

exchange

-LRB-

SAX

-RRB-

solid

phase

extraction

column

-LRB-

mg

Varian

Associates

USA

-RRB-

and

washed

through

with

deionized

water

-LRB-

ml

-RRB-

The

eluate

was

discarded

and

the

fraction

containing

both

mono

and

bisphosphates

of

the

sugars

was

eluted

with

M

-LRB-

ml

-RRB-

and

collected

in

a

polypropylene

centrifuge

tube

-LRB-

ml

-RRB-

Most

of

the

buffer

was

removed

by

blowing

the

sample

dry

with

a

stream

of

dry

nitrogen

gas

whilst

warming

the

sample

in

a

water

bath

at

°C

The

samples

were

then

dephosphorylated

using

human

prostatic

acid

phosphatase

which

was

chosen

for

its

ability

to

catalyze

dephosphorylation

quantitatively

without

causing

the

transformations

produced

by

other

phosphatases

-LRB-

Irvine

et

al.

-RRB-

Dephosphorylation

of

up

to

μmoles

of

sugar

phosphates

was

carried

out

by

the

addition

of

units

of

prostatic

acid

phosphatase

to

the

sugar

phosphate

solution

in

M

ammonium

acetate

acetic

acid

buffer

pH

-LRB-

typically

μl

-RRB-

and

incubated

for

h

at

°C

The

reaction

was

terminated

by

heating

at

°C

for

min

Following

dephosphorylation

the

solutions

containing

the

free

sugars

were

deionized

by

stirring

for

min

with

g

of

mixed

bed

resin

prepared

using

equal

weights

of

anion

-LRB-

Bio-Rad

AG

mesh

form

-RRB-

and

cation

-LRB-

Bio-Rad

AG

W

mesh

H

form

-RRB-

exchange

resins

together

with

deionized

water

The

resin

was

removed

by

filtration

through

a

small

column

fashioned

from

a

pipette

tip

-LRB-

ml

-RRB-

plugged

with

cotton

wool

and

containing

a

further

g

of

the

mixed

bed

resin

The

sample

was

washed

through

with

water

-LRB-

ml

-RRB-

collected

in

a

polypropylene

centrifuge

tube

-LRB-

ml

-RRB-

and

lyophilized

The

resulting

residue

was

washed

with

deionized

water

-LRB-

ml

-RRB-

into

an

Eppendorf

tube

-LRB-

ml

-RRB-

and

again

lyophilized

The

residue

was

redissolved

in

water

-LRB-

μl

-RRB-

and

the

entire

sample

was

transferred

to

a

Reacti-Vial

-LRB-

μl

-RRB-

and

dried

over

The

sugars

were

subsequently

dried

under

high

vacuum

for

h.

Derivatization

of

dephosphorylated

sugar

phosphates

Sugars

were

prepared

for

GC/MS

analysis

as

the

methoxime-TMS

derivatives

using

methods

similar

to

those

first

described

by

Sweeley

-LRB-

Sweeley

et

al.

-RRB-

for

mg

scale

derivatizations

In

our

investigation

smaller-scale

derivatizations

were

required

Up

to

nmol

-LRB-

approx.

μg

-RRB-

of

the

dried

sugars

in

Reacti-Vials

-LRB-

ml

-RRB-

were

derivatized

by

the

addition

under

dry

nitrogen

of

methoxylamine

in

pyridine

-LRB-

μl

-RRB-

and

refluxing

at

°C

for

h

in

a

Reacti-therm

heater

The

mixture

was

cooled

to

room

temperature

then

silylation

was

accomplished

by

the

addition

under

dry

nitrogen

of

μl

of

Regisil

-LRB-

Pierce

-RRB-

followed

by

heating

for

h

at

°C

The

mixture

was

cooled

to

room

temperature

then

diluted

as

appropriate

with

dry

pyridine

for

GC/MS

analysis

Injections

of

μl

ideally

contained

no

more

than

pmol

of

the

sugar

under

analysis

in

order

to

avoid

saturation

of

the

mass

selective

detector

as

determined

by

sensitivity

runs

with

derivatized

glucose

GC/MS

analysis

of

derivatized

sugars

Derivatized

sugars

were

analyzed

by

capillary

GC/MS

on

a

Hewlett

Packard

gas

chromatograph

interfaced

with

a

Hewlett

Packard

MSD

and

controlled

by

a

Hewlett

Packard

ChemStation

Gas

chromatography

was

performed

on

an

capillary

column

-LRB-

m

mm

i.d.

μm

cross-linked

methyl

silicone

stationary

phase

-RRB-

in

the

splitless

mode

with

helium

carrier

gas

-LRB-

flow

rate

ml

min

-RRB-

The

column

temperature

was

held

at

°C

for

min

with

the

filament

off

for

the

passage

of

the

solvent

and

excess

reagent

through

the

column

The

temperature

was

then

increased

to

°C

at

°C

min

and

held

at

°C

for

min

Spectra

were

obtained

by

electron

impact

-LRB-

EI

-RRB-

ionization

at

eV

Full

scan

spectra

of

the

derivatized

sugars

were

usually

obtained

by

scanning

from

m/z

to

at

a

rate

of

scans

s

and

represent

an

average

of

scans

taken

across

the

top

of

each

relevant

chromatographic

peak

Selected

ion

monitoring

-LRB-

SIM

-RRB-

experiments

on

clusters

of

ions

of

interest

in

the

spectra

of

the

derivatized

sugars

were

carried

out

in

groups

of

ions

per

peak

with

dwell

times

of

ms

per

ion

All

results

represent

the

mean

of

at

least

three

separate

injections

unless

otherwise

stated

Corrections

for

natural

abundance

isotopes

of

C

H

N

O

and

Si

and

contributions

from

adjacent

ions

one

mass

unit

lower

of

comparable

intensity

were

made

in

calculating

enrichments

in

individual

ions

Only

fragment

ions

which

had

been

found

to

have

essentially

a

single

origin

as

the

result

of

the

GC/MS

analysis

of

specifically

sugar

standards

were

used

in

this

study

-LRB-

Irvine

et

al.

MacLeod

et

al.

-RRB-

Identification

of

sugar

phosphates

in

chloroplast

extracts

The

individual

unlabelled

sugar

phosphates

present

in

the

chloroplast

extracts

were

identified

by

the

retention

times

and

full

scan

mass

spectra

of

their

dephosphorylated

Mox-TMS

derivatives

on

the

above

GC/MS

system

when

compared

with

those

of

authentic

sugars

run

under

identical

conditions

-LRB-

Irvine

et

al.

MacLeod

et

al.

-RRB-

Quantification

by

GC/MS

of

sugar

phosphates

in

chloroplast

extracts

Quantification

of

analytes

is

best

achieved

by

the

inclusion

in

the

sample

mixture

of

a

known

amount

of

an

internal

standard

having

similar

chemical

and

physical

characteristics

to

those

of

the

analytes

Ideally

for

GC/MS

analysis

the

internal

standard

should

be

a

stable

isotope

analogue

of

the

analyte

under

investigation

and

when

there

are

multiple

analytes

a

stable

isotope

of

each

analyte

is

preferred

In

this

investigation

however

the

analytes

-LRB-

derivatized

sugars

-RRB-

are

themselves

expected

to

become

multiply-labelled

with

the

isotope

during

photosynthesis

and

the

stable

isotope

dilution

method

therefore

could

not

be

used

The

alternative

approach

was

used

of

adding

a

compound

that

is

similar

to

the

sugars

under

investigation

but

is

unlikely

to

be

encountered

in

chloroplasts

and

would

be

well

separated

from

the

analytes

on

GC/MS

For

SIM

the

mass

spectrum

of

the

derivatized

standard

chosen

for

this

GC/MS

investigation

needed

to

contain

an

ion

which

was

common

to

all

of

the

sugars

being

measured

In

addition

this

ion

must

not

become

labelled

in

a

experiment

The

sugar

alcohols

seemed

to

be

an

obvious

choice

The

mass

spectra

of

the

per-TMS

derivative

of

these

compounds

show

a

prominent

m/z

ion

representing

the

pentamethyldisiloxonium

ion

-LRB-

-LSB-

-LRB-

-RRB-

-LRB-

-RRB-

-RSB-

-RRB-

as

do

the

mass

spectra

of

all

of

the

sugar

methoxime

TMS

derivatives

Since

this

ion

does

not

contain

any

carbons

derived

from

the

sugar

it

can

not

become

labelled

during

a

isotope

incorporation

experiment

Thus

SIM

of

the

m/z

ion

was

the

method

of

choice

for

quantitation

studies

Erythritol

was

chosen

as

the

internal

standard

because

-LRB-

-RRB-

the

m/z

ion

of

its

per-TMS

derivative

gave

a

linear

and

reproducible

response

when

calibrated

against

the

same

ion

in

the

mass

spectra

of

Mox-TMS

sugars

at

levels

up

to

pmol

and

-LRB-

-RRB-

it

was

well

separated

on

the

GC

column

from

the

sugars

under

consideration

Sugars

which

were

available

in

the

free

form

with

a

high

degree

of

purity

were

dried

for

days

over

phosphorous

pentoxide

under

a

low

vacuum

Approximately

g

of

the

dried

powder

or

syrup

-LRB-

ketopentoses

-RRB-

was

weighed

to

five

decimal

places

to

prepare

standard

solutions

of

each

sugar

Appropriate

dilutions

of

these

were

taken

to

prepare

a

set

of

mM

standard

solutions

Some

sugars

were

only

available

as

the

phosphate

esters

and

frequently

in

the

presence

of

various

amounts

of

buffer

salts

In

these

cases

after

weighing

the

dried

powders

and

preparing

standard

solutions

they

were

assayed

enzymically

to

establish

their

concentrations

prior

to

use

Sedoheptulose

D-glycero

D-ido

and

D-glycero

D-altro-octuloses

were

assayed

as

their

bisphosphates

using

an

aldolase-based

assay

-LRB-

Bergmeyer

and

Bernt

-RRB-

Erythrose

was

assayed

as

the

monophosphate

ester

using

a

transketolase-based

assay

-LRB-

Paoletti

et

al.

-RRB-

Sugar

phosphates

-LRB-

μmol

-RRB-

were

then

evaporated

to

dryness

in

a

rotary

film

evaporator

and

dephosphorylated

in

a

solution

-LRB-

ml

-RRB-

which

contained

citrate

buffer

μmol

pH

-RRB-

and

human

prostatic

acid

phosphatase

-LRB-

units

-RRB-

After

allowing

the

dephosphorylation

to

proceed

for

h

at

°C

the

reaction

was

terminated

by

heating

at

°C

for

min

The

samples

were

then

treated

with

mixed

bed

resin

and

lyophilized

Recovery

from

the

dephosphorylation

step

was

assessed

by

carrying

out

parallel

dephosphorylations

of

glucose

fructose

and

fructose

all

of

which

were

assayed

enzymically

as

the

free

sugars

The

recovery

of

the

monophosphates

was

and

of

the

bisphosphate

was

These

figures

provided

a

useful

estimate

of

the

likely

recoveries

of

those

sugars

-LRB-

above

-RRB-

which

could

not

be

assayed

in

the

non-phosphorylated

form

Appropriate

dilutions

of

the

lyophilized

sugars

were

then

prepared

to

produce

a

set

of

mM

solutions

for

use

as

standards

Groups

of

sugar

mixtures

were

prepared

each

containing

μl

of

the

mM

standard

solution

Each

sugar

was

thus

present

in

a

volume

of

ml

and

at

a

concentration

of

mM

The

first

group

contained

erythrose

fructose

and

D-glycero

D-ido-octulose

The

second

group

contained

glucose

sedoheptulose

and

D-glycero-D-altro-octulose

Pentoses

were

calibrated

separately

A

series

of

dilutions

of

the

standard

mixtures

containing

or

nmol

of

each

sugar

together

with

nmol

of

the

erythritol

standard

were

added

to

separate

ReactiVials

The

mixtures

were

then

first

dried

over

then

under

high

vacuum

and

derivatized

-LRB-

Mox-TMS

-RRB-

This

gave

solutions

which

contained

or

pmol

of

each

sugar

together

with

pmol

of

the

erythritol

standard

in

each

μl

of

the

derivatized

solution

GC/MS

analysis

was

carried

out

by

the

measurement

of

the

peak

areas

obtained

for

the

m/z

ion

of

each

sugar

using

single

ion

monitoring

Three

injections

were

made

at

each

sugar

level

and

the

average

ratio

of

sugar

to

erythritol

peak

area

was

determined

In

those

cases

where

the

Mox-TMS

derivative

of

the

sugar

showed

peaks

for

both

syn

and

anti-forms

the

area

of

the

leading

peak

was

used

A

graph

plotting

the

measured

ratio

of

each

sugar

m/z

ion

area

to

the

erythritol

m/z

ion

area

against

the

actual

ratio

of

the

sugar

to

erythritol

for

the

series

of

sugar

concentrations

was

then

plotted

The

slopes

of

these

graphs

then

represent

the

calibration

factors

relating

the

sugars

to

the

internal

standard

erythritol

and

were

determined

as

the

slope

of

the

least

squares

line

of

regression

of

measured

area

ratio

on

actual

ratio

-LRB-

Table

-RRB-

Table

calibration

factorsaSugarInterceptSlopeCoefficient

of

correlation

-LRB-

r

-RRB-

Erythrose

Xylulose/Ribulose

Fructose

Glucose

Sedoheptulose

D-a-Octulose

D-i-Octulose

aErythritol

used

as

internal

standard

Results

and

discussion

Initial

experiments

were

carried

out

using

the

leaf-disc

oxygen

electrode

during

the

time

that

the

special

photosynthesis

apparatus

was

being

constructed

in-house

Obvious

disadvantages

inherent

in

this

approach

were

-LRB-

-RRB-

the

difficulty

in

achieving

complete

instantaneous

displacement

of

with

and

-LRB-

-RRB-

the

time

taken

to

dissemble

the

apparatus

and

quench

the

photosynthesis

reaction

after

exposure

of

the

chloroplasts

to

leaving

open

the

possibility

of

partial

back

exchange

of

the

sugars

One

advantage

was

that

the

photosynthetic

activity

of

the

chloroplast

preparations

could

be

measured

in

situ

prior

to

carrying

out

the

exchange

experiments

There

were

several

pitfalls

that

had

to

be

overcome

before

PS

exchange

experiments

with

could

be

performed

Foremost

of

these

was

the

low

concentrations

of

sugars

of

the

PS

pathway

observed

in

the

chloroplast

extracts

in

initial

experiments

using

unlabelled

together

with

the

presence

of

other

sugar-like

components

in

the

GC/MS

analysis

The

latter

was

eventually

traced

to

adsorption

and

decomposition

of

the

small

quantities

-LRB-

pmol

-RRB-

of

sugar

phosphates

present

in

the

chloroplasts

on

the

surfaces

of

glass

vessels

used

during

the

workup

procedure

To

minimise

decomposition

these

were

replaced

where

possible

with

non-glass

vessels

Losses

due

to

adsorption

could

not

be

completely

avoided

and

necessitated

combining

extracts

from

five

consecutive

experiments

-LRB-

using

the

same

chloroplast

preparation

-RRB-

in

order

to

try

to

obtain

useful

data

on

those

sugars

present

in

lowest

concentrations

Contaminants

introduced

during

the

workup

presented

another

problem

in

the

GC/MS

analysis

which

required

extensive

investigation

of

their

sources

The

only

contaminant

that

could

not

be

completely

eliminated

was

sorbitol

but

this

did

not

interfere

with

the

sugar

analysis

as

the

retention

time

of

its

TMS

derivative

differed

from

those

of

the

Mox-TMS

sugars

under

investigation

The

experiment

carried

out

in

the

oxygen

electrode

at

zero

time

exposure

of

the

chloroplast

preparation

to

in

nitrogen

showed

after

work-up

no

incorporation

of

label

in

the

sugars

As

well

the

control

experiment

carried

out

with

chloroplasts

exposed

to

in

the

dark

for

s

produced

no

enrichment

in

any

of

the

sugars

in

the

chloroplast

extract

After

analysis

of

the

results

of

four

experiments

carried

out

in

the

oxygen

electrode

involving

exposure

of

the

chloroplasts

to

for

s

-LRB-

twice

-RRB-

and

for

s

-LRB-

twice

-RRB-

only

one

of

the

s

experiments

produced

a

reasonably

comprehensive

set

of

results

-LRB-

Table

-RRB-

The

main

factor

contributing

to

the

inadequacy

of

the

remaining

experiments

was

the

lack

of

sufficient

material

to

enable

GC/MS/SIM

runs

to

be

performed

on

all

of

relevant

ion

clusters

as

set

out

in

a

previous

paper

-LRB-

see

Table

MacLeod

et

al.

-RRB-

Table

resultsa

for

s

experiment

No.

in

the

leaf

disc

oxygen

Unidentified

aAfter

correction

for

natural

abundance

of

and

contributions

from

some

neighbouring

ions

Results

represent

an

average

of

at

least

three

consecutive

GC/MS

runs

Standard

deviations

for

most

sugars

were

and

for

the

less

abundant

sugars

-LRB-

xylose

arabinose/lyxose

octulose

and

unidentified

heptulose

-RRB-

standard

deviations

were

When

significantly

different

enrichments

were

measured

for

the

syn

and

anti-methoxime

isomers

it

was

assumed

that

some

interference

was

present

causing

the

higher

result

and

the

one

showing

the

lower

enrichment

was

used

otherwise

both

syn

and

anti-isomers

where

present

were

used

in

the

calculationsbLyxose/Arabinose

refers

to

either

one

or

both

sugars

which

could

not

be

separated

on

the

capillary

GC

column

used

in

these

analysescRibose/Ribulose

refers

to

both

sugars

which

although

they

could

not

be

resolved

were

both

shown

to

be

present

from

their

characteristic

mass

spectra

-LRB-

MacLeod

et

al.

-RRB-

dThe

intensity

of

the

m/z

ion

is

enhanced

due

to

column

bleed

despite

background

correctioneThe

MS

of

Ribose

contains

a

small

m/z

ion

while

that

of

Ribulose

has

a

large

m/z

ionfThe

m/z

ion

contributes

to

m/z

and

No

correction

has

been

made

for

this

Experiments

were

then

carried

out

using

the

custom-made

photosynthesis

apparatus

involving

exposure

of

isolated

chloroplasts

to

in

for

s

in

the

dark

and

for

and

s

under

controlled

light

conditions

After

work-up

both

the

dark

experiment

and

zero

time

exposure

showed

no

incorporation

in

any

of

the

sugars

analysed

As

was

the

case

for

all

but

one

of

the

oxygen

electrode

experiments

the

s

and

s

experiments

after

work-up

did

not

contain

a

sufficient

concentration

of

the

sugar

Mox-TMS

derivatives

even

after

combining

five

experiments

to

enable

selective

ion

profiles

to

be

obtained

on

all

of

the

relevant

ions

in

all

of

the

sugars

This

was

particularly

disappointing

as

the

specially

designed

apparatus

performed

well

otherwise

In

all

of

the

above

experiments

the

concentrations

of

the

sugar

phosphates

present

in

the

chloroplast

extracts

were

determined

by

SIM

of

the

m/z

ion

present

in

the

mass

spectra

of

the

dephosphorylated

Mox-TMS

derivative

using

erythritol

as

internal

standard

-LRB-

see

Materials

and

methods

-RRB-

A

correction

was

made

for

losses

incurred

during

the

chloroplast

work-up

and

dephosphorylation

stages

by

adding

glucose

to

a

single

membrane-mounted

chloroplast

preparation

immediately

prior

to

quenching

in

the

special

apparatus

The

measured

recovery

figure

of

of

glucose

represents

a

minimum

recovery

value

since

the

method

eventually

used

involved

the

pooling

of

the

extracts

from

five

membranes

which

would

be

expected

to

lead

to

reduced

losses

Table

lists

the

values

obtained

As

expected

the

two

blank

runs

showed

only

traces

of

glucose

fructose

and

pentoses

while

the

s

dark

experiment

showed

lower

than

normal

levels

of

all

sugars

except

glucose

The

other

six

experiments

five

using

the

oxygen

electrode

and

one

using

the

special

apparatus

showed

sugar

concentrations

which

although

varying

from

experiment

to

experiment

were

of

the

same

order

of

magnitude

to

those

previously

reported

-LRB-

Lilley

et

al.

-RRB-

Table

metabolite

concentrations

-LRB-

nmol

-LRB-

mg

chl

-RRB-

-RRB-

Equipment

for

experimentApparatusLeaf

disc

oxygen

electrodeExposure

to

-LRB-

sec

-RRB-

.2933.710.312.6729.6623.4842.06117.658.43

Unidentified

heptulose0.000.000.000.000.290.000.851.040.84Sedoheptulose0.019.040.011.0612.971.1721.9538.9016.26D-g

daExtract

containing

chloroplast

medium

without

s

exposure

to

in

the

darkcTentatively

identifieddm/z

ion

not

monitored

No

sugars

could

be

analysed

under

the

GC

conditions

used

since

they

eluted

with

the

residual

derivatizing

agents

A

tetrose

sugar

was

present

which

had

the

same

mass

spectrum

and

elution

time

as

erythrose

-LRB-

Table

-RRB-

but

subsequent

analysis

of

label

incorporation

into

selected

ions

showed

no

enrichment

The

unidentified

heptulose

listed

in

Table

did

show

very

significant

incorporation

in

the

few

ion

clusters

measured

-LRB-

Table

-RRB-

comparable

to

that

observed

in

sedoheptulose

D-g-D-a-Octulose

was

identified

by

the

retention

times

of

its

syn

and

anti-isomers

but

the

earlier

eluting

D-g-D-i-octulose

was

not

detectable

The

enrichment

results

obtained

in

Table

were

sufficient

to

calculate

atom

excess

in

some

but

not

all

of

the

individual

carbon

atoms

of

the

sugars

listed

-LRB-

see

Table

in

MacLeod

et

al.

-RRB-

For

example

the

enrichment

in

of

ribose

-LRB-

-RRB-

was

obtained

by

subtraction

of

m/z

-LRB-

-RRB-

from

m/z

-LRB-

-RRB-

Similarly

subtracting

the

enrichments

in

the

ions

m/z

and

-LRB-

-RRB-

gave

the

atom

excess

in

-LRB-

-RRB-

was

calculated

by

subtracting

the

above

value

for

from

the

enrichment

in

the

m/z

ion

-LRB-

-RRB-

In

some

cases

where

it

was

not

possible

to

obtain

enrichment

data

for

individual

carbon

atoms

values

for

consecutive

two

carbon

units

were

calculated

While

it

is

obvious

that

the

results

obtained

fall

short

of

the

objective

for

reasons

given

above

they

nevertheless

allow

some

pertinent

observations

to

be

made

The

presence

of

D-g-D-a-octulose

in

isolated

chloroplasts

at

concentrations

between

and

nmol

-LRB-

mg

chl

-RRB-

is

unequivocally

confirmed

-LRB-

Table

-RRB-

In

the

s

experiment

the

atom

excess

in

carbons

and

of

the

octulose

is

between

and

comparable

with

the

values

calculated

for

and

of

ribose

and

of

fructose

and

glucose

and

and

of

sedoheptulose

-LRB-

Table

-RRB-

These

sugars

and

their

above

carbons

are

implicated

in

fixation

in

Calvin

's

photosynthetic

pathway

-LRB-

Fig.

-RRB-

and

it

is

therefore

not

unreasonable

to

speculate

that

D-g-D-a-octulose

also

has

a

role

in

this

pathway

in

support

of

the

conclusions

drawn

in

the

preceding

paper

-LRB-

Williams

and

MacLeod

-RRB-

The

pattern

of

labelling

of

carbons

and

of

D-g-D-a-octulose

presented

here

is

precisely

the

same

as

that

shown

in

the

octulose

by

NMR

spectroscopy

of

a

spinach

leaf

extract

after

photosynthesis

in

-LRB-

Bartlett

et

al.

-RRB-

Table

excess

in

individual

carbons

and

enrichments

in

moietiesaCarbonXyloseLyxoseXyluloseRiboseFructoseGlucoseSedoheptuloseUnk

-LCB-

-LCB-

-LCB-

c

-LCB-

d

-LCB-

d

-LCB-

-LCB-

b

c

d

-LCB-

d

-LCB-

dash

implies

that

the

value

could

not

be

measured

as

a

one

or

two-carbon

unitbCalculated

from

two

separate

sets

of

data

e.g.

minus

and

minus

both

give

a

value

for

of

XylosecNot

corrected

for

contribution

of

m/z

to

the

m/z

clusterdm/z

from

residual

column

bleed

taken

as

zero

in

m/z

cluster

A

further

interesting

observation

was

the

presence

of

a

second

heptulose

sugar

albeit

in

much

lower

concentrations

than

that

of

sedoheptulose

-LRB-

Table

-RRB-

It

had

a

substantial

atom

excess

at

of

comparable

with

that

observed

for

the

same

carbon

in

sedoheptulose

A

GC/MS

comparison

of

the

retention

time

of

its

Mox-TMS

derivative

with

that

of

mannoheptulose

eliminated

this

as

a

possibility

and

so

it

remains

unidentified

One

of

the

anomalies

of

the

Calvin

Pathway

-LRB-

Fig.

-RRB-

has

been

the

failure

to

identify

erythrose

phosphate

in

chloroplasts

even

though

it

is

proposed

to

play

an

integral

role

in

fixation

In

this

study

although

we

were

able

to

detect

a

four

carbon

sugar

with

the

same

retention

time

and

mass

spectrum

as

Mox-TMS-erythrose

-LRB-

Table

-RRB-

GC/MS/SIM

analysis

showed

no

incorporation

of

in

any

of

its

fragment

ions

It

could

therefore

be

assumed

that

the

level

of

erythrose

phosphate

participating

in

the

pathway

is

below

that

detectable

by

GC/MS/SIM

However

the

identification

and

evidence

for

the

presence

of

Ery

in

the

Calvin

and

Pentose

pathways

has

always

been

weak

and

contentious

-LRB-

Williams

et

al.

-RRB-

Unfortunately

circumstances

have

not

allowed

us

to

repeat

the

above

experiments

on

a

scale

which

could

provide

sufficient

amounts

of

sugar

phosphates

to

allow

more

complete

data

on

the

time-course

of

incorporation

from

during

photosynthesis

to

be

obtained

In

support

of

an

earlier

study

of

gluconeogenesis

in

liver

cells

-LRB-

Desage

et

al.

-RRB-

it

has

however

been

shown

here

that

GC/MS/SIM

offers

a

viable

alternative

methodology

for

the

study

of

positional

isotope

analysis

of

intermediates

formed

from

stable-isotope

labelled

precursors

in

complex

biological

systems

In

summary

the

results

reported

here

and

in

the

preceding

paper

have

established

the

formation

and

specific

predictive

labelling

of

octulose

phosphate

-LRB-

s

-RRB-

in

spinach

chloroplasts

by

and

during

photosynthesis

The

data

do

not

unequivocally

prove

that

octulose

phosphates

are

direct

and

exclusive

intermediates

of

a

new

set

of

partial

reactions

for

the

path

of

carbon

that

were

inadvertently

missed

by

Calvin

and

his

colleagues

However

we

suggest

that

octulose

phosphate

formation

may

fulfil

a

role

as

mono

and

bisphosphates

in

a

shunt

-LRB-

as

defined

by

Newsholme

and

Leech

-RRB-

that

is

attached

to

the

Calvin

reaction

scheme

-LRB-

Fig.

-RRB-

Figure

is

a

structural

display

of

the

new

reaction

scheme

presented

in

the

accompanying

paper

The

shunt

shows

that

the

octulose

phosphate

reactions

intersect

with

and

may

in

time

and

space

compartmentalize

Seh

from

SBPase

and

thereby

limit

its

important

role

in

the

regulation

of

flux

in

the

carbon

path

of

PS

Fig.

extension

of

the

Calvin

pathway

by

the

inclusion

of

an

octulose

phosphate

shunt

-LRB-

vide

infra

-RRB-

Each

asterisk

-LRB-

-RRB-

represents

a

carbon

atom

labelled

during

one

turn

of

the

cycle

Sugar

phosphates

in

red

represent

those

participating

in

the

octulose

shunt

For

explanation

of

abbreviations

see

preceding

paper

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chloroplasts
octulose phosphate shunt
selected ion monitoring

13c-enriched sugar phosphates
13co2 incorporation
gas chromatography–mass spectrometry
modified calvin cycle
mox-tms derivatives


Purinergic signaling microenvironments : An introduction The common theme of this introductory article and the minireviews that follow in this special issue is the concept of microenvironments within tissues and surrounding cells that would be ideal signaling venues for a biologically active purinergic ligand . Collectively , the editors/authors and the other contributing authors agree that nucleotides and nucleosides would be most potent within a confined system . A talented cadre of purinergics has been solicited to discuss purinergic signaling in his or her favorite microenvironment within a given organ or tissue . We are gratified by the large number of original articles that also have successfully navigated the peer review process and are part of this special issue . These concepts are not simply purinergic , but the idea of maximal potency in a tissue microenvironment and surrounding specialized cells within a tissue pertains to any autacoid or paracrine agonist . Introduction Adenosine 5 ′ triphosphate -LRB- ATP -RRB- and adenosine are extracellular autocrine and paracrine signaling molecules . The most potent effects of ATP and adenosine are likely limited to extracellular microenvironments within tissues or immediately surrounding a cell or group of cells . These extracellular microenvironments may have a limited area of diffusion , have a small volume , have an ideal ionic composition and pH , represent an unstirred layer near a membrane surface , represent a trapped interstitium , or any and all of the above . If P1 or P2 nucleotide receptors are present on a membrane that is exposed to released nucleotide or nucleoside in this microenvironment , these ligands can engage P1 G-protein-coupled receptors , P2Y G-protein-coupled receptors or P2X receptor channels and affect the function of the cell on which it binds . Besides ATP and adenosine , other forms of nucleotides and nucleosides may also be active extracellular signaling ligands and may have unappreciated receptors . Inosine , thymidine , and even cyclic nucleotides are released by and may have effects on mammalian cells . The self-aggregation signal for the slime mold Dictyostelium discoideum , is cyclic adenosine monophosphate -LRB- AMP -RRB- . Receptors for cyclic nucleotides have only been defined until now in the lower biological systems . Receptors for cyclic nucleotides as well as inosine or thymidine may also exist on mammalian cells and receive this autocrine signal within a microenvironment . Ideal microenvironments for purinergic signaling For simplicity , we focus on ATP and its metabolites . Many different microenvironments have been studied in great detail and are described and included below . One classical microenvironment that has emerged in importance in the cystic fibrosis -LRB- CF -RRB- research community is in an open system but is a stirred liquid layer trapped between the luminal membrane of the airways epithelium and the humidified air that fills the lung and airways . The airway surface liquid -LSB- ASL , also termed airway surface fluid -LRB- ASF -RRB- , the periciliary liquid layer -LRB- PCL -RRB- , and the epithelial lining fluid -LRB- ELF -RRB- -RSB- bathes motile respiratory cilia -LSB- 1 -- 4 -RSB- . The humidified air above this layer and the ciliated apical or luminal membrane beneath this layer help close this microenvironment . Submucosal gland secretions may also contribute importantly to its composition . Ric Boucher and his many coworkers have presented many recent seminal contributions on the purinergic signaling that may occur in the ASL -- among its many other properties -LSB- 3 , 4 -RSB- . There are similar microenvironments of this type that are also discussed in review articles within this special issue . Daniel Marcus and colleagues and Claire Mitchell and coworkers discuss liquid microenvironments in the ear and the eye , respectively -LSB- 5 , 6 -RSB- , which provide a medium for purinergic signaling . A second classical microenvironment is a closed and open system that is compromised of a secretory gland or acinus and a collecting or draining duct . Iva Novak and colleagues have done seminal work in the pancreas in this regard -LSB- 7 , 8 -RSB- . Geoff Burnstock has often discussed the rich purinergic signaling that occurs in tubes or ducts -LSB- 9 , 10 -RSB- . In this microenvironment , glandular and acinar secretions may convey the purinergic ligands so that they may act in an autocrine manner at the acinus or in a paracrine manner within the duct . However , it is an open system in that these secretions eventually exit this tissue environment . The airway submucosal gland beneath the airway surface epithelium may be one such example that conveys purinergic signals to the surface where they may drive ciliary beat and other functions . The pancreatic acinus and duct , the sweat gland and duct , and the bile secretory apparatus and the bile duct are all important examples . A third microenvironment is a closed system in which the purinergic ligands concentrate within a small volume and within a tissue . Here , the purinergic ligands bind and activate receptors until they are either degraded or reabsorbed by the cells that secreted them or by neighboring cells . The endolymph of the ear and the humors of the eye are ideal examples of such closed systems -LSB- 11 , 12 -RSB- . Marcus and colleagues and Mitchell and coworkers discuss these systems in minireviews alluded to above -LSB- 5 , 6 -RSB- . Original articles and reviews in this issue and future issues of Purinergic Signalling speak to such systems in pancreatic islets and in the many renal tubules that comprise the nephron of the kidney -LSB- 13 -RSB- . In polycystic kidney and liver disease , tubules remodel into abnormal fluid-filled cysts . Multiple laboratories have argued that the fluid-filled lumen of the encapsulated cyst is a rich yet pathophysiological microenvironment for purinergic signaling -LSB- 14 -- 20 -RSB- . The minireviews that follow this introductory article focus on specific microenvironments , cells and tissues that create and/or come in contact with such microenvironments , and all possible modes of purinergic signaling within these microenvironments . An ideal microenvironment paradigm for extracellular purinergic signaling : the synapse Many reviews in the past -LSB- 21 -- 23 -RSB- and in the initial issues of this journal have documented well the roles of the P2X receptor channel at the synapse between neurons . Indeed , P2Y receptors may play roles in synaptic transmission as well . This is analogous to the interplay between cholinergic and muscarinic receptor systems in cholinergic neurons and synapses in many different nuclei and regions of the brain . In fact , the purinergic field was born from the classical studies of extracellular ATP on the heart by Drury and Szent-Gyorgyi -LSB- 24 -RSB- , in the definition of purinergic nerves in the enteric nervous system by Burnstock and colleagues -LSB- 25 -RSB- , and on the release and effects of purinergic ligands such as ATP on skeletal muscle contractility by Tom Forrester -LRB- see `` Historical Perspective '' by T. Forrester in this issue , -LSB- 26 -RSB- -RRB- . The authors lead this series of minireviews with the idea that the synapse is the ideal and , perhaps , the first or founding microenvironment for autocrine and paracrine purinergic biology . The synapse is designed to have a limited diffusion distance between pre - and postsynaptic membranes where the principal ligand or neurotransmitter is reabsorbed by the presynapse or degraded in the synapse . For the sake of simplicity , our narrative here focused first on a purinergic synapse where released ATP acts as the principal ligand to evoke an action potential in the postsynaptic membrane . ATP as the lead neurotransmitter is released in response to an action potential in the presynaptic bulb that opens voltage-dependent calcium -LRB- Ca2 + -RRB- channels . This voltage-dependent Ca2 + entry signal triggers the fusion or exocytosis of ATP-filled synaptic vesicles so that labile ATP is secreted into the synaptic cleft . Extracellular ATP-gated P2X receptor channels on the postsynaptic membrane have a large extracellular domain poised to bind that ATP immediately upon release within a synaptic microenvironment where there is a short diffusion distance and a small volume . In other words , the synapse is an ideal microenvironment for paracrine purinergic signaling . In this paradigm , the ATP is likely degraded to adenosine diphosphate -LRB- ADP -RRB- , 5 ′ AMP and adenosine , where adenosine may be recycled back into the presynapse by nucleoside transporters only to be a substrate to synthesize new ATP for metabolism or neurotransmission . Several additional functions for P2X receptor channels have been proposed in neurotransmission driven by other neurotransmitters . For example , at the glutamate synapse , it is well known that ATP is released as a cotransmitter . A classical example of this is the hair-cell sound transduction process , where glutamate and ATP are coreleased to stimulate auditory afferents -LSB- 11 -RSB- . How may ATP affect responses to glutamate at glutamate , N-methyl-D-aspartate -LRB- NMDA -RRB- , and kainate receptor channels in the postsynaptic membrane and/or at the presynapse in terms of release of ligand ? Many possible cotransmitter effects have also been entertained and studied . We do not wish to debate the relative roles of P2X receptor channels in neurotransmission . The possible explanations of ATP effects at an excitatory glutamatergic synapse are given in simplistic terms . We only wish to show the synapse as a lead microenvironment to illustrate how and why local ATP signaling may be important . First , the presence of a postsynaptic P2X receptor channel or channels would potentiate the excitation of glutamate postsynapse . Not only do NMDA , kainate , and glutamate itself potentiate Ca2 + and sodium -LRB- Na + -RRB- entry as Ca2 + permeable nonselective cation channels , but extracellular ATP-gated P2X receptor channels open a separate pathway for Ca2 + and Na + . Flow of Ca2 + and Na + through parallel pathways driven by these cotransmitters would lead to a much quicker development of a postsynaptic potential . Moreover , with Ca2 + entering with Na + , it is likely that the action potential may be more long-lived and possibly more robust -LRB- i.e. , having a shoulder or plateau due to Ca2 + entry beyond the Na + - driven spike -RRB- . Second , ATP could potentiate glutamate release at the presynaptic neuron by potentiating Ca2 + entry driven by the preceding action potential . A more robust Ca2 + entry would cause more or secondary glutamate release and would keep the postsynaptic neuron firing . As for acetylcholine , ATP also has G-protein-coupled receptors , the P2Y receptors , which could modulate presynaptic and/or postsynaptic neuron function . Third , could metabolites of ATP limit activation of the postsynaptic and presynaptic neuron ? One does not wish for the postsynaptic neuron to continue firing ad infinitum . One must have a limiting or negative feedback signal . Could this signal be adenosine ? Not only does adenosine need to be recycled back into the neuron as a substrate for new ATP synthesis , but adenosine may also bind to P1 G-protein-coupled receptors to have limiting effects or neuronal function . Of course , ATP release and signaling at an inhibitory synapse driven by γ-aminobutyric acid -LRB- GABA -RRB- or glycine would have different and modulating effects on inhibitory synapse . We do not discuss or illustrate these possible mechanisms because that is not the intent of this introductory minireview . Synaptic purinergic neurotransmission has been reviewed by Burnstock and others in many seminal reviews -LSB- 9 , 10 , 21 -- 23 -RSB- . Conclusions and future directions The founding concepts of purinergic signaling within tissues and tissue microenvironments were found in the heart , the enteric system -LRB- later to be duplicated in urinary bladder afferents , nociceptive neurons at the dorsal root ganglia of the spinal cord , etc. -RRB- , and skeletal muscle by Forrester and coworkers -LSB- 26 -RSB- . This special issue of Purinergic Signalling focusing on the `` Physiology of Nucleotide Release '' is dedicated to these and other pioneers who would not give up the fight . All purinergics would agree that we owe the purinergic pioneers a great debt . Through their efforts , establishment of this journal , and the increasing volume of research in purinergic signaling , it is now accepted that ATP and its metabolites have two principal biological roles -- as an extracellular signaling ligand and as an intracellular fuel .

O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O B O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O B O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B O O O O O O O

adenosine atp nucleosides nucleotides signaling