STRUCTURE OF GLYCOGENS AND AMYLOPECTINS

II. ANALYSIS BY STEPWISE ENZYMATIC DEGRADATIOS*

BY JOSEPH LARNER,t BARBARA ILLINGWORTH, GERTY T. CORI, AND CARL F. CORI

(From the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri)

(Received for publication, June 6, 1952)

Three models have been proposed for the structure of branched polysac- charides of the starch-glycogen class; these are illustrated in Fig. 1. Staud- inger and Husemann (1) proposed a comb-like structure in which each side branch arises from a single main branch. Haworth (2) favored a laminated structure in which each branch arises from a preceding branch. Finally, Meyer et al. (3) proposed an irregular, tree-like structure; they pointed out that the action of p-amylase on a polysaccharide with a laminated structure would result in the formation of an elongated, thread-like molecule, as indicated in Fig. 1. Acetates of such molecules yield elastic films on evaporation of a solution on a glass plate. The acetylated fl-amylase limit dextrins of amylopectin and glycogen did not form films and were therefore assumed to have a more compact structure than is compatible with the Haworth model.

In the present experiments, four branched polysaccharides (rabbit muscle and liver glycogen, corn and wheat amylopectin) were degraded 80 to 00 per cent by separate and successive action of phosphorylase and amylo- 1,6-glucosidase. The values found after each enzymatic step could be made to fit only one model; namely, the one representing the four polysac- charides as multibranched, tree-like structures.

EXPEKIMENTAL

Glycogen was isolated from livers and muscles of several well fed rabbits by the method described in Paper I (4). The amylopectins were kindly supplied by Dr. R. W. Kerr.

Muscle phosphorylase was recrystallized eight to nine times and was

* This work has been supported in part by a research grant from the Corn Indus- t,ries Research Foundation.

t Postdoctorate Fellow of the National Institutes of Health, United States Public Health Service, 1948-50. This report is from a dissertation submitted in partial fulfilment for the degree of Doctor of Philosophy in Biological Chemistry, Washing- ton University. Part of this research was carried out during the tenure of a Life Insurance Medical Research Fellowship.

641

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642 STEPWISE DEGRADATION OF GLYCOGEN

shown to be free of glucosidase by the test previously described (5). The glucosidase preparations (5) after dialysis for 18 hours contained from 3 to 5 y of inorganic P per ml. They were treated with dry starch powder (5) in order to remove cr-amylase. Different batches of U. S. P. corn-starch varied in their ability to remove a-amylase, and even with the same batch there was some variation in the effectiveness of this treatment with different muscle extracts. For these reasons each glucosidase preparation was tested for amylase activity under conditions duplicating those used in the degrada- tion experiments. A reaction mixture containing 12 mg. of glycogen and 3 ml. of starch-treated extract was incubated at 30” for 4 hours, depro- teinized according to West (see Steiner (6)), and copper reduction deter-

I 2 3

1- I I *

1 I’

,’ . , I _ . , , % ,’ I R R R

FIG. 1. Models proposed for the structure of glycogen or amylopectin, (1) Staud- inger, (2) Haworth, (3) Meyer. The dotted Ibnes indicate the limits of degradation by @-amylase. R is the reducing group.

mined in the filtrate. Only those extracts were used in which the increase in reduction (in terms of glucose) corresponded to a splitting of less than 1 per cent of the added glycogen. That such traces of amylase did not interfere seriously in the degradation experiments was indicated by the fact that glucosidase action reached an end-point after 2 to 3 hours of incu- bation, as shown by determinations of the reaction product, glucose, by copper reduction, and by a specific microenzymatic method.’ The subse- quent phosphorylase step was also uninfluenced, since identical amounts of glucose phosphate were released after treatment with glucosidase prepara-

1 Although the action of cr-amylase leads to an increase in reducing power, there is little or no glucose liberated until degradation has progressed quite far (7). Traces of amylase may thus interfere with the chemical but not with the microenzymatic determination of the glucose formed by glucosidase action. Good agreement between chemical and enzymatic glucose determinations serve as a further check for the effec- tive removal of amylaae from the glucosidase preparation.

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LARKER, ILLISGWORTH, CORI, AND CORI 643

tions which were practically free of amylase activity and preparations which had a slight, but measurable, amylase activity. It should be noted that glucosidase does not act on glycogen, but only on the limit dextrin of glycogen produced by preceding phosphorylase action.

Stepwise Enzymatic Degradation-Starting with 150 to 200 mg. of poly- saccharide, five to seven successive digestions in which the two enzymes were alternated were carried out. The products formed after each enzy- matic step were determined quantitatively as follows: After phosphorylase action, glucose-l-phosphate was determined in HgClz-HCl filtrates by cop- per reduction after acid hydrolysis for 5 minutes at 100” (5). After glu- cosidase action, free glucose was determined in HgC&-HCl filtrates by the specific enzymatic-spectrophotometric method2 (5), and in some cases also by the micromethod of Park and Johnson (8) with satisfactory agreement.

The details of the degradation experiments were as follows: Phospho- rylase was allowed to act first in a medium containing about 15 moles of inorganic phosphate per mole of glucose calculated to be available to the enzyme in the formation of glucose-l-phosphate. When the reaction had reached an end-point (usually after 2 to 3 hours at 30”), the remaining polysaccharide was isolated by precipitation with alcohol and in some cases again submitted to the action of phosphorylase. There occurred no or only an insignificant further degradation. The material obtained by this exhaustive treatment with phosphorylase was designated as limit dex- trin, or LD,; it was isolated and freed of inorganic phosphate by carrying out several alcohol precipitations, one of them at pH 4 to 5. The ab- sence of phosphate in the final product was checked by analysis.

LD1 was next incubated with 250 units of glucosidase (5) per mg. of polysaccharide (in a medium to which no phosphate was added) until two successive periods 1 hour apart showed no further liberation of free glucose; the total period of incubation was usually 3 hours at 30”. Glucosidase was inactivated by heating at 100” for 5 minutes and the coagulated protein removed by filtration. Phosphorylase and inorganic phosphate were again added and the incubation continued until the enzymatic action had reached an end-point, as judged by the absence of further liberation of glucose-l- phosphate in two successive incubation periods 1 hour apart. This resulted in the formation of LD2, which was isolated as indicated above.

A repetition of the process yielded LD,, and in some cases also LDI. Use of larger amounts of polysaccharide initially would have permitted

* Owing to the presence of traces of inorganic phosphate and of phosphorylase in the glucosidase preparations, a small amount of hexose phosphate, usually less than 10 per cent of the free glucose, was also formed. This was determined spectrophoto- metrically with the Zwischenferment system before the addition of adenosinetri- phosphate.

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644 STEPWISE DEGRADATION OF GLYCOGEN

further degradation. The amount of polysaccharide recovered at each LD stage was usually 80 to 95 per cent of theory.3 End-group determina- tions were done on aliquots of the isolated LD by means of the enzymatic method previously described. As a further aid in the elucidation of struc- ture, some LD preparations were also submitted to the action of crystalline p-amylase, kindly supplied by Dr. A. K. Balls.

Results

The analytical values obtained on stepwise enzymatic degradation are presented in Table I. Repeated determinations of the per cent degradation in the first phosphorylase step were in excellent agreement. Less satis- factory results were at first obtained with the glucosidase step. In the trial run of this series, which was carried out on liver glycogen (Table I), it was not appreciated that the reaction had failed to reach completion within the limited period of incubation (kept brief in order to avoid amylase action). That an incomplete glucosidase step can reduce the extent of subsequent degradation by phosphorylase and thus produce an accumula- tive error is shown by comparing the first and second series of degradation of liver glycogen. This difficulty was avoided in subsequent determina- tions; compare the two separate degradations of muscle glycogen to the LD2 stage.

Omitted from Table I are data on the degradation of the various LD preparations by P-amylase. The outer structure of LD1 (phosphorylase) has been illustrated (5); originating from a branch point there is a main branch of 6 to 7 glucose units in 1,4 linkage, and a side branch of 1 glucose unit in 1,6 linkage, the latter being the point of attack of glucosidase. LD1, when treated with /3-amylase, yields 2 maltose units from each of the main outer branches. The same is true for LD2 and LDI and they all have therefore the same outer structure. The inner structure of LD1, LD2, and LD3 also appears to be a repetition of a fairly regular pattern. Thus t,he per cent end-group and per cent degradation by phosphorylase of glucosidase-treated LDi, LDZ, and LD3 of liver glycogen, second series, remains almost constant. The other polysaccharides show a slight de- crease in the per cent end-group between LDr and LD, and some variation in the per cent degradation by phosphorylase, pointing to a somewhat greater irregularity of inner structure.

In Table I values are presented in terms of per cent degradation of the original material after each phosphorylase step. As pointed out before (4)) neither phosphorylase nor P-amylase can remove all the 1,4-linked glucose residues in the outer chains down to the first branch point. By

* For quantitative determination, aliquots of the polysaccharides were hydrolyzed in acid and determined as glucose.

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TABL

E I

Slep

wise

De

grad

ation

of

Po

lysac

char

ides

by

Phos

phor

ylase

an

d Am

ylo-1

,6-g

lucos

idase

The

value

s ar

e giv

en

in

per

cent

de

grad

ation

(o

r pe

r ce

nt

end-

grou

p)

of

the

mat

erial

in

dica

ted

at

the

head

of

ea

ch

colum

n.

Re-

sults

for

tw

o se

para

te

degr

adat

ions

of

liver

gly

coge

n ar

e sh

own,

th

e fir

st

one

being

th

e tri

al

run

of

the

serie

s (s

ee

the

text).

Pe

r ce

nt

degr

adat

ion

of

the

orig

inal

m

ater

ial

w&s

calcu

late

d fro

m

aver

ages

of

th

e va

lues

show

n,

exce

pt

for

liver

gly

coge

n,

in

which

va

lues

from

th

e se

cond

se

ries

only

were

us

ed.

LD

= lim

it de

xtrin.

- I-

I- LD

z LD

a LD

I

I Gg

estec

lig

ested

by

glu

- :os

idasc

y

phos

- ho

rylas

t

I ,

-

Origi

nal

Degr

ada-

tio

n of

origin

al m

ateria

l En

d- gro

up

Cges

ted

i ph

os.

olylas

I

I 1

e

$;=f

;- ori

ginal

mate

rial

30.8

37.9

40.7

41

.1

61.6

12.4

2.

1

12.0

4.

0

10.3

4.

0

33.0

34

.6

37.0

40.0

76.8

42.0

63

.8

79.1

44.5

40.0

71.1

2.

8 3.

3 32

.8

81.2

8.7*

4.

3 5.

1

53.1

73.2

88

.0

1

-

Polys

acch

aride

En

d- gro

up

12.8

12.0

10.6

‘iges

ted

BY

lu-

4 0s

as

e

Snd-

roup

6.8

6.8

Xges

tec

by p

hos

horyl

as

36.2

34.8

End-

3roop

2.5

2.7

3.2

5.6

6.3

4.6

5.1

5.3

6.5

7.3

4.8

5.5 6.1

4.7 5.3

I e ’

Rabb

it liv

er

glyco

gen

11.7

11.3

10.9

10.2

2.3

30.8

33

.8

4.3

44.0

87

.5

“ m

uscle

gly

coge

r

Whe

at

amylo

pecti

n

Corn

“

* No

t in

clude

d in

th

e ca

lcula

tion.

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646 STEPWISE DEGRADATION OF GLYCOGEN

calculation, if the glucose residues of outer branches were completely re- moved in the formation of LDI, the two glycogens would be degraded 61

per cent, wheat amylopectin 70 per cent, and corn amylopectin 74 per cent. Liver glycogen after the fourth treatment with phosphorylase, correspond- ing to the formation of LDr, was degraded 87.5 per cent. If the outer chains of LD4 had been completely removed, the degradation would have been over 90 per cent. This residual structure near the reducing end is still a large molecule, since the original molecular weight of glycogen is of the order of 1 million or more according to ultracentrifugation and osmotic pressure measurements.

After each phosphorylase step one “tier” has been degraded and the next tier is made accessible through action of glucosidase and so on. The question arises how many such tiers are present in the different polysac-

TABLE II

Length oj Outer and Inner Branches and Distribution of Branch Points

Polysaccharide

Rabbit liver glycogen “ muscle “

Wheat smylopectin Corn ”

No. of residues per outer branch Average No. of

A;;;;; hydrolyzed glucose residues* Branch points in

length successive tiers, .- per cent

Outer ‘hyqsaPsl- ~8-hy1ase branches b::%s

* The method of calculation is described in the preceding paper (4).

charides and how are they arranged. Inspection of the three proposed models in Fig. 1 shows that the Staudinger and the Haworth models would yield a constant percentage of the total branch points in each tier during successive enzymatic degradation, while the Meyer model would yield a diminishing percentage as one progresses from the outer to the inner tiers, such as is actually indicated by the data in Table II. Only one kind of model could be made to fit this arrangement of branch points; namely, one which represents the polysaccharides as multibranched, tree-like structures.

Fig. 2 represents a model of a segment of rabbit muscle glycogen (mol. wt. 24,000) in the form of a branched structure, containing five tiers and constructed according to the following specifications: average chain length 15, average length of outer and inner branches 9 and 6 glucose residues, respectively, 50 per cent of branch points in the first tier, 20 per cent in the second tier, and 10 per cent each in the remaining three tiers. These are close to the experimental values found in Table II. and they put a consid- erable restriction on the type of model that can be constructed. In fact, the significance of the analytical data can be tested by comparing experi-

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LARNER, ILLINGWORTH, CORI, AND CORI 647

FIG. 2. Model of segment of muscle glycogen based on results obtained by step- wise enzymatic degradation. 0, 8, and 8 glucose residues removed by first, second, and third degradation with phosphorylase, respectively. 0, glucose resi- dues removed by amylo-l,6-glucosidase. Of five tiers three were degraded, corre- sponding to 122 out of 150 glucose residues.

TABLE III Experimental and Calculated Values fOT Model of Segment of Glycogen Molecule from

Rabbit Muscle

The calculated values are those resulting from the construction of the mode1 in Fig. 2. LD refers to the limit dextrin formed by phosphorylase and LD(g) to the limit dextrin after treatment with glucosidase. The total number of glucose residues is 150, of which 140 are in a-l,4 and 10 also in (Y-I ,6 linkage, the latter being the resi- dues at branch points.

Stage of degradatior

Glycogen “

LD, LD, LDlk) LD2 LDs LDdg) LDs

I liE

i-

-

nzyme used for degradation Tgzdof

Phosphorylase a-l,4

Glucosidase a-1,6 Phosphorylase a-1,4

Glucosidase I a-1,6 Phosphorylase a-1,4

Degradation

55 36.6 34.8

5 5.3 5.4 40 44.5 41.3

2 4.0 4.0 20 41.6 40.0

End-group

Ca’cu- Found lated 1

cr cent per cc?&1

6.7 6.8

10.5 11.3

10.0 10.3

10.7 10.6

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A48 STEPWISE DEGRADATION OF GLYCQGEN

mental and calculat,ed values, the latter being those result,ing from the con- struction of the model according to the above specifications. This has been done in Table III. As an example, if it is assumed that the first tier c*ont,ained 40 per cent of the branch points (instead of 50 per cent,), t,hc per WII~ cud-group c~nkulatctl for the l,D, would be I I .8 (instead of 10.0). It can be seen that the experimental value of 10.3 is much closer to the latter value. Good agreement between calculated and experimental values has also beeu obtained with the other three polysaccharides, aud the models which have been constructed for them are very similar to that shown in Fig. 2.

DISCUSSION

Consideration of the mechanism of enzymatic synthesis of polysaccha- rides permits certain deductions to be made about structure. It has been shown that straight chains of 4 or more glucose units can act as primers for the reaction catalyzed by potato phosphorylase. Polysaccharide syn- thesis from glucose-l-phosphate consists in the addition of glucose units to the non-reducing end of these chains, which grow in a regular fashion, as shown by the gradual change in the iodine absorption spectrum corre- sponding to visual colors from red to purple to blue (9). It has been found recently that, when there is added to this system a purified fraction of branching enzyme from liver, the polysaccharide synthesized has the char- acteristics of glycogen or amylopectin. This will be reported in a later paper. The quest.ion here is what kind of structure would result; two possibilities are illustrated in Fig. 3, A and B. It is assumed in Fig. 3, A that the new end-groups created by branching all have the same affin- ity for phosphorylase, and hence there is’ an equal chauce for any of the branched chains to be made longer. This would result in the forma- tion of a tree-like structure similar to that in Fig. 2. If this were not the case, that is, if one branch had a higher affinity than the others, a laminated structure would result, as shown in Fig. 3, B.

A distinction between these two possibilities is afforded by the kinetics of the reaction. With low primer concentration, the curve for polysac- charide synthesis with phosphorylase plus branching enzyme has been shown to be S-shaped (lo), a fact which has been verified in recent observa- tions. This is due to the fact that phosphorylase is far from saturated with respect to primer end-groups and that the type of branrhing illustrated iu Fig. 3, A gives rise to a rapid increase in primer concentration. Formatioii of a laminated structure (Fig. 3, R) would not give an autocatalytic curve, because the number of effective primer end-groups in each moleceule being built up would remain the same.4

4 Evidence for the action of the branching enzyme on a structure growing as in Fig. 3, A has been obtained (11). The outer chains were made longer with CL’-glucose.

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LARNER, ILLINGWORTH, CORI, AND CORI G49

The structure in Fig. 3, A shows the following characteristics. As the number of successive tiers increases, the per cent of the total number of branch points in the outermost tier approaches 50 per cent, in the next 25

FIG. 3. Schematic representation of combined action of phosphorylase and branch- ing enzyme. A represents the growth of the molecule when all chains have an equal chance of being made longer by phosphorylase. In B only one of the branched chains is made longer by phosphorylase action.

TABLE IV

Calculated Molecular Weights for Glycogen with 6.8 and Corn Amylopeclin with 4.1 Per Cent End-Groups

The calculations are based on the model inFig. 3, A and are as follows: the number of outside branches equals the number of end-groups, the latter being 6.8 per cent for glycogen. Therefore, sixteen end-groups correspond to a total of (16 X 100)/6.8 = 235 glucose residues, or (X 162) to a molecular weight of 38,ooO.

No. of outside branches

16 4 38,cOo 63,100 32 5 76,000 126,500 ‘64 6 152,000 253,000

512 9 1,216,OOO 2,020,ooo

No. of tiers __-- 6.8 per cent end-group 4.1 per cent end-group

per cent, in the next 12.5 per cent, and so on. l’his complete regularity of st,ructure represents an ideal case; as a fairly close approach the model for

The polysaccharide was isolated and the branching enzyme alone allowed to act on it. A shifting of chain segments, as illustrated, would then result in the formation of 1,6-linked Cl’-labeled glucose units. This could be demonstrated by means of am- ylo-1,6-glucosidase, which is specific for these units.

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650 STEPWISE DEGRlDATION OF GLYCOGEN

muscle glycogen in Fig. 2 yielded 50, 20, and 10 per cent of the branch points in the first, second, and third tiers, respect.ively.

The relationship between structure and molecular weight may now be discussed. In Table IV are calculated values for the model in Fig. 3, A for 6.8 and 4.1 per cent end-group, corresponding to glycogen and corn amylopectin, respectively. It is of interest to note how strongly the mole- cular weight is influenced by structure. The structure shown in Fig. 3, A when built up to five tiers has a molecular weight of 76,000.5 A lami- nated model of muscle glycogen with five tiers, as in Fig. 3, B, would have a molecular weight of only 14,300.

Glycogen and amylopectin are polydisperse substances, as indicated by ultracentrifugation. If the mean molecular weight were of the order of 1 million or more, glycogen would contain an average of nine tiers according to Table IV, and stripping it of its four outermost tiers would be equivalent to a degradation of over 90 per cent, such as has actually been reached in the case of liver glycogen. The analytical data are compatible with a molecule of this size. In fact, the method of enzymatic degradation as used does not give much information about molecular size, because the total number of tiers present cannot be accurately determined. By calcu- lation, a remainder of about 7 per cent of an original molecule of 1 million would contain five tiers and would have a molecular weight of 76,000. The aim is to isolate this fragment in order to determine the details of structure and the number of tiers near the reducing end.

SUMX4RY

Stepwise enzymatic degradation of two glycogens and two amylopectins was carried out by alternate action of muscle phosphorylase and amylo- 1,6-glucosidase. The limit dextrins formed by phosphorylase action were isolated. After the third phosphorylase step liver glycogen was degraded 77 per cent and corn amylopectin 89 per cent. An additional cycle brought the degradation of liver glycogen to 88 per cent. The successive phos- phorylase limit dextrins were similar in structure, as shown by the fact the /3-amylase removed in each case 2 maltose units per main outer branch and that the per cent end-group remained fairly constant. The per cent of the total branch points present in each tier became progressively less as the reducing end was approached. Only one type of model could be made to fit the analytical data; namely, one which iepresents the polysaccharides as multibranched, tree-like structures. Further support for this structure arises from a consideration of the mechanism of enzymatic synthesis.

6 The model in Fig. 2 differs from that in Fig. 3, A in two respects. The sequence of branching from inner to outer tiers is haphazard rather than according to a pat- tern of regular growth, aa in Fig. 3, A. Secondly, a segment of a molecule is repre- sented rather than the whole molecule.

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LARNER, ILLINGWORTH, CORI, AND CORI 651

BIBLIOGRAPHY

1. Staudinger, H., and Husemann, E., Ann. Chem., 627, 195 (1937). 2. Haworth, W. N., Chem. and Znd., 17, 917 (1939). 3. Meyer, K. H., Bernfeld, P., Boissonnas, R. A., Gurtler, P., and Noelting, G.,

J. Phys. and Colloid Chem., 33, 319 (1949). 4. Illingworth, B., Larner, J., and Cori, G. T., J. Biol. Chem., 199, 631 (1952). 5. Cori, G. T., and Larner, J., J. Biol. Chem., 188, 17 (1951). 6. Steiner, A., Proc. Sot. Exp. Biol. and Med., 33, 968 (1935). 7. Pazur, J. H., French, D., and Knapp, D. W., Proc. Iowa Acad. SC., 67, ‘203 (1950). 8. Park, J. T., and Johnson, M. J., J. Biol. Chem., 181, 149 (1949). 9. Swanson, M. A., J. Biol. Chem., 173,825 (1948).

10. Cori, G. T., and Cori, C. F., J. Biol. Chem., 161, 57 (1943). 11. Larner, J., Federation Proc., 11, 245 (1952).

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Cori and Carl F. CoriJoseph Larner, Barbara Illingworth, Gerty T.

DEGRADATIONSTEPWISE ENZYMATIC

AMYLOPECTINS: II. ANALYSIS BY STRUCTURE OF GLYCOGENS AND

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