BIOCHENJSTRY 101

AUTUMN QUARTER 1961

In recognition of and in the spirit of the Honor Code, I certify that I will

neither receive or give unpermitted aid and that I will report to the best of my
ability all Honor Ccde violations observed by me.

Questions 1 through 7:

An infant was recently referred to the Pa10 Alto-Stanford Hospital with a

diagnosis of possible glycogen storage disease of the von Gierke type.  Following
the preliminary examination and laboratory work-up, the case was presented at
one of the weekly pediatric conferences,  To begin with, a comparison was made
between the characteriDtics of classical von Gierke's disease and those found in
the present case; these are summarized in the accompanying table.  The opinion
of those present at the conference was unanimous; although this infant displayed
massive deposition of glycogen and a marked enlargment of the liver, there was
sufficient reason to doubt the original diagnosis,  Moreover, there was general
agreement that a correct diagnosis required measurements of the level of various
enzymes involved in glycogen synthesis and utilization together with an analysis
of the structuse of the liver glycogen,  Mr, Harbandray, one of the first-year
medical students who was present at the conference, saw in this an opportunity
for an it3tercsting research problem and he volunteered to obtain this information,

1,     To confirm the tentative conclusion that this infant did not have classical
van Gierke's disease, he obtained a liver sample by biopsy and tested for (choose
the one which is Zefective in von Gferke's disease) arzd found it to be present in
amounts within the normal range :

(a) phosphorylase - b
(b) gfucokirase
(c ) glu.co se 6-pho sphatase
(a) amylo 1,6 glucosidase

2,
one of several enzymes might be absent or deiective.  %e of the iollowing groups
list3 the enzymes wbich haw in ca?nr,ion the feakrz that a deiect in zuy one of the
enzymes would lead to the negative ai?serralin reapornse.

The fact that the response ta adrezih was xiegztiv3 suggested that any

Choose that group:

(a)

(b ) glucose -6 - pho s2kata de hgdrogeaase, amylase, phospho hemseiso -

(c) UDPG-glycogen synthetase, phosphorylase I a

(d) arnylo- 1,4 -> 1,6 transglucosylase, amylo- 1,6 glucosidase,

51-AMP-cyclase, phosphorylzae b kinaDe, amylo- 1,b glucosidase,

phosphorylase - a alad phosphogiucor&tsse


merase and aldolase

phosphoglucomutase

CEanoaldolase and transkc tolaoe

phosphorylase - a and UDPG-glycogen synthetase.

TABLE FOR QUESTIONS 1-27

Glycogen Level

Markedly elevated
in liver but normal
in muscle

Disease

I

Blood Sugar Sugar Response to Glycogen
                            Adrenalin Structure

Level Tolerance *

          Norma? to glucose;
Very low abnormal to galac- Blood sugar Normal
            tose and fructose level

No rise in

vcn Gierke's

I

Markedly elevated
in liver but normal
in muscle

Present
patient

I

Normal to glucose,
galactose and blood sugar Not done
fructose level

No zise in

Normal to
sligh%ly low

I I

1

9

Rate of disappearance Qf injected or ingested sugar from circulating blood,

3.
normal amountso  As a result of these findings, Mr. Harbandray felt that some
clue to identifying the defect would emerge from an examination of the glycogen
structureo
phosphate and amylo- 1,6 glucosidase should be degraded completely to :

(a) a mixture of glucose-1-phosphate and sucrose
    11 I1 I1 11 It glucose 6-phosphate
    I1 tI I1 I1 11 glucose

(b) ''

      I1 I1 11 II I1 maltose
(4 I'
(dl I'

Again he found that each of the enzymes tested was present in essentially

He knew that normal glycogen incubated with purified phosphorylase E,

4;
isolated from the liver biopsy yielded only glucose4 -phosphate,  Since the amount
of glucose-1-phosphate formed wa s not very large but rather about what one would
have expected from the action of phosphorylase a alone on normal liver glycogen,
he concluded that the glycogen contained :

He observed, however, that in the test performed above the glycogen

(a) excessively short outer chains
(b) excessively long outer chains
(c) normal outer chains
(d) nothing but a 1,6 glucosyl linkages

5.    From his experiments thus far Nire Harbandray
a defect in the glycogen structure and not in the enzymes

reasoned that there was
which degrade it. hiore-

over, he realized that the aberrant structure must result from a defect in one of
the enzymes involved in glycogen synthesis.
probably not in UDPG-glycogen synthetase,  He recalled, moreover, that a loss
of amylo-l,4 -> 1,6 transglucosylase activity would result in the formation of :

He argued that the defect was

(a) a glycogen with a greater than normal proportion of branches
(b) a glycogen with a normal number of branches
(c) a glycogen with no branches
(d) a glycogen containing only e-l,6 glucosyl linkages

63    Even though the existence of such a defect was not wholly consistent with
his finding in #4, he decided to go ahead and assay the extracts from the patient's
liver biopsy for amylo- 1,4 --> 1,6 trans2lucosylase activity in the following way.
The C14-labaled glycogen substrate uaed in the assay was prepared from CI4-
glucose-1-phosphate, unlabeled glyccgen as primers and phosphorylase a.
incubating this labeled glycogen with normal arnylo- 1,4 -> 1,6 transgluczsylase
lie expected that subsequent exposure of the product to phosphorylase - a and
phosphate would remove :

(a) all the C -glucose residues as glucose-1-phosphate
(b) only a portion of the CL4-glucose residues but these would all be
(c) part of the C14 as glucose-1-phosphate and the remainder as glucose
(d) nothing

Upon

14

glucose- 1-phosphate

7.
much to his surprise he found essentially normal levels of transglucosylase
activity. He was faced therefore with the dilemma of rationalizing how in the
presence of normal amylo-l,4 -> 1,6 transglucosylase, a glycogen was formed
which could not be split by the combined action of phosphorylase and amylo-l,6
glucosidase, Realizing that something might be strange about the inter-glucosyl
linkages being formed with the patient's enzyme, he asked one of his graduate
student friends in the chemistry department to determine the type of linkages
between glucose residues in a sample of the infant's liver glycogen;  Back came
the answer that the predominant linkages wexe 1,4, some were 1,3, but no 1,6
linkages, Mr, Harbandray saw the answer immediately; the defect in glycogen
structure which prevented it from being metabolized was due to:

When he assayed extracts from the patient's liver by this technique,

(a) a modified UDPG-synthetase which formed 1,3 instead of 1,4 linkages
(b) an altered amylo-l,6 glucosidase which could split 1,3 linkages
(c) the suspected amylo 1,4 -> 1,6 transglucosylase which was in reality

  an amylo 1,4 -> 1,3 transglucosylase
(d) a modified phosphorylase - a which could not phosphorolyze 1,3 linkages

   For his ingenious solution to this case Mr. Harbandray was awarded the
Job A, Berg research medal and this &isease came to be known as Harbandray's
syndrome.

8,
transformations except :

Succinyl CoA has been found to undergo all of the following enzymatic

(a) hydrolytic deacylation
(b) GOA transfer to acetoacetate
(c) isomerization to form methyl malonyl GOA
(d) fixation of C02 to form a ketoglutaryl CoA

tt

9,
stearyl thiokinase, stearyl GOA, AMP and pyrophosphate are formed in equimolar
amountse
Which of the following experimental observations supports the proposed role of
stearyl adenylate as an intermediate :

When ATP, CoA,stearic acid and Mg

are incubated with the enzyme

One proposal is that stearyl adenylate is an intermediate in this reactiono

32

(a) the radioactivity of P -labeled pyrophosphate will exchange with

(b) the radioactivity of P32-labeled pyrophosghate will exchange with

ATPin the pzesence of enzyme, Mgtt and stearateb

ATP in the presence of enzyme, Mgtt and CcA,

(c) C14-labeled AMP will exchange with ATP in the presence of enzyme,
  M and stearate.,
(a) C"-labeled AMP will exchange with ATP in the presence of enzyme,
Mett and GOA,