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by Gerty T. Cori and Barbara IllingworthS. Nussenbaum, W. Z. Hassid and With a note 

AMYLOPECTIN

ENZYMATIC SYNTHESIS OF

ARTICLE:

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ENZYMATIC SYNTHESIS OF AMYLOPECTIN*

BY S. NUSSENBAUM AND W. Z . HASSID(From the Division of Plant Biochemistry, Colle ge of Agriculture, University

of California, Berkeley, California)

WITH A NOTE BY GERTY T. CORI AND BAR BAR A ILLINGWORTH

(Rece ived for publication, January 29, 1951)

The problem of branching in glycogen and starch has been the subject

of interest ever since Cori and Cori (I, 2) isolated the phosphorolytic

enzyme from animal tissues and Hanes (3) found a similar enzyme inplants. Examination of polysaccharides produced by these enzymes when

glucose-l-phosphate was used as a substrate showed that, similar to amyl-

ose, they had a linear configuration in which the glucose units are com-

bined through 1,4-glucosidic linkages only (4, 5). However, naturally

occurring glycogen and most starches also contain 1,6 linkages. It was

therefore assumed that synthesis in vivo of glycogen and of the amylopectin

starch component requires two enzymes and that in the process of prepa-

ration of the muscle or potato phosphorylase the enzyme producing the1,6 linkages is eliminated. Proceeding on this assumption, Cori and Cori

(6) produced evidence for the existence of a branching factor in liver and

heart, capable of synthesizing 1,6 linkages. The combined action of the

branching enzyme and crystalline muscle phosphorylase resulted in the

formation of a polysaccharide which close ly resembled glycogen. Later,

Haworth, Peat, and Bourne (7) reported the isolation from potatoes of an

enzyme, the so called Q enzyme, which, in association with potato phos-

phorylase, catalyzed the synthesis of amylopectin from glucose-l-phos-

phate.

In a series of papers, Peat and his collaborators (8-14) presented evi-

dence that the Q enzyme converts linear amylose into branched amyl-

opectin. Since this reaction proceeds without the presence of inorganic

phosphate, they hold the view that Q enzyme functions as a non-phospho-

rolytic enzyme. Their evidence is based chiefly on the following experi-

mental facts. When Q enzyme is allowed to act upon amylose, a product

is produced that gives a purple-red color with iodine; the synthetic material

is soluble in water, and does no,t retrograde from solution; it is attacked byP-amylase with the liberation of maltose, but hydrolysis is arrested before

conversion is completed; end-group analysis by the methylation method

indicates the existence of chains of about 20 glucose units in average

length (8).

* Supported in part by a grant from the Corn Industr ies Research Foundation.

673

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674 ENZYMATIC SYNTH ESIS OF AMYLOPECTIN

However, Beckmann and Roger (15) were not able to duplicate these

results. They claim that the material obtained by the English investi-

gators is not amylopectin, but is an artifact. According to their findings,potato preparations contain fatty acids which form a complex with amyl-

ose, possessing many of the properties of amylopectin. They therefore

concluded that these properties have been erroneously used by Haworth,

Peat, and their associates as evidence to prove that their product is amylo-

pectin.

Recently Bernfeld and MeutBmddian (16-19) reported the isolation from

potatoes of an enzyme “isophosphorylase,” which appeared to resemble

the Q enzyme, inasmuch as it presumably played a part in the formationof amylopectin from both amylose and glucose-l-phosphate. There is,

however, a difference between the action of the two enzymes. Whereas

inorganic phosphate is not, involved in the formation of branched poly-

saccharide by the Q enzyme, the Swiss investigators claim that phosphate

constitutes an integral part of the branching mechanism. They believe

that isophosphorylase catalyzes reversibly the formation of 1,6 bonds in a

reaction analogous to that of the formation of 1,4 bonds by phosphorylase.

The evidence offered by Bernfeld and Meutem dian for the synthesisof amylopectin is as follows: When a mixture of isophosphorylase and

phosphorylase acts on amylose in the presence of inorganic phosphate, a

product is formed which gives a purple color with iodine and is degraded

by /3-amylase to the extent of 65 per cent, while amylose is degraded by

this enzyme to the extent of 100 per cent. A similar product is obtained

when a mixture of these enzymes acts on glucose-l-phosphate. Isolation

and further characterization of the synthetic product were not undertaken.

In view of these conflicting results, it, was of interest to reinvestigate the

problem of enzymatic synthesis of amylopectin. The conversion of amyl-

ose to amylopectin with Q enzyme, reported by Peat and his associates

( 14), was successfully repeated, and further chemical and biochemical

evidence was obtained showing that the synthetic product is branched.

Like amylopectin, the synthetic material is precipitated with dilute alco-

hol, produces a purple color with iodine, and gives a very small reducing

value. On treatment with amylase a hydrolysis limit of 51 per cent is

obtained. End-group determination by periodate oxidation shows an

average of 21 glucose residues per end-group, while with an enzymaticmethod a value of 20 is obtained. Estimation of the molecular weight by

osmotic pressure measurements of the acetylated product indicates a value

of 54,000, calculated as the deacetylated product. In contrast to amylose,

the synthetic material is ‘as effective a “primer” of muscle phosphorylase

as corn amylopectin.

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S. NUSSENBAUM AND W. Z . HASSID 675

The synthesis of amylopectin with Bernfeld and Meutemedian’s (16-19)

“isophosphorylase” could not be repeated. Treatment of amylose with a

potato preparation made according to these authors resulted in a degra-dation product which gave a purple color with iodine, but which on further

analysis proved to be a mixture of short chain linear dextrins.

Of the two mechanisms proposed for amylopectin synthesis, that based

on the work of Peat and his collaborators appears to be well substantiated.

The Q enzyme is apparently capable of splitting 1 ,4-glucosidic linkages in

the amylose chain and exchanging them for 1,6 linkages without the medi-

ation of phosphate, thus forming branches. Like the enzymes present in

Pseudomonas saccharophila (20), Leuconostoc mesenteroides (21), and Neis-seria per vu (22), the Q enzyme can thus be regarded as belonging to the

class of trans-glycosidases.

EXPERIM ENTAL

Preparation of Q Enzyme-The enzyme preparations were made accord-

ing to the procedure described by Barker, Bourne, and Peat (11). Batches

of approximately 5 kilos of potatoes yielded 2 liters of crude juice. The

method involves precipitation from potato juice of Q enzyme and other

proteins by the addition of lead acetate at pH 7.25, elution of the lead-

protein complex with sodium hydrogen carbonate solution, through which

a stream of carbon dioxide is passed, and precipitation of the enzyme from

the supernatant liquid with neutral ammonium sulfate. The final solution

was diluted to 400 ml. and kept in the refrigerator at 2’. The preparation

was analyzed for inorganic phosphate (23) and was found to contain 0.0035

mg. per ml. of solution.

Conversion of Amylose to Amylopectin-A 3 gm. sample of corn amylose

was dissolved in 0.5 N sodium hydroxide, diluted to 1300 ml., and neutral-

ized with 0.5 N sulfuric acid. Then 500 ml. of Q enzyme and 500 ml. of

1 M acetate buffer, pH 7.0, were added, and the mixture was diluted to

3000 ml. The digest was allowed to remain at room temperature for 24

hours, and during the incubation period aliquot portions were analyzed

for reducing sugars by the method of Hassid et al. (24, 25). The reducing

values of a control experiment in which amylose was omitted were simul-

taneously determined. The difference between the reducing values of the

two digests was considered a measure of the reducing groups liberated bythe action of the enzyme preparation on amylose. Different preparations

differed in the amount of reducing substances formed, but none showed

an increase in 24 hours greater than 1.5 per cent (calculated as maltose)

of the amylose added.

The digest was heated, the coagulated proteins removed by filtration,

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676 ENZYMATIC SYNTH ESIS OF AMYLOPECTIN

and the solution dialyzed for several days until the dialysate was practi-

cally free of iodine-staining material’ and inorganic impurities. The solu-

tion was then concentrated in vacua at 60” and the material precipitatedwith ethanol. It was dried in a vacuum oven at 50” and extracted with

methanol in a Soxhlet extractor for 12 hours to eliminate fatty acid im-

purities and dried again. A yield of 55 per cent of synthetic amylopectin

was obtained, based on the original amylose. The product produced a

purple-red color when treated with iodine and was soluble in water.

Hydrolysis with /3-Amylase-The method used for hydrolysis of the syn-

thetic polysaccharide with P-amylase was essentially that of Bernfeld and

Giirtle r (26), except that the maltose produced was estimated by oxidationwith ferricyanide (24, 25). The crystalline p-amylase (27) was obtained

from Dr. A. K. Balls of Western Regional Research Laboratory.

A 25 mg. sample of the polysaccharide was dissolved in 20 ml. of water.

A solution containing an excess of P-amylase, approximately 500 units

(28), and 6 ml. of acetic acid-sodium acetate buffer, pH 4.8, was added.

The mixture was diluted to 50 ml. and 5 ml. aliquots were periodically

drawn during 24 hours for determination of reducing values. It was

found that the synthetic amylopectin was hydrolyzed to the extent of 51per cent.

A 20 mg. sample of amylose originally used for the amylopectin synthesis

was dissolved in 2 ml. of 2 N sodium hydroxide, neutralized with hydro-

chloric acid, diluted to 20 ml., and similarly treated with p-amylase. The

hydrolysis limit of the amylose with this enzyme was 90 per cent. Further

addition of , amylase did not increase the extent of hydrolysis of either

the synthetic amylopectin or the amylose.

Fatty Acid Content of Synthetic Polysaccharic le-A 0.2 gm. sample of the

material was hydrolyzed completely by boiling under a reflux condenser

with 15 ml. of 3 N sulfuric acid for 1 hour. The solution was extracted for

24 hours with ether in a Soxhlet extractor and the ether extract was shaken

twice with 5 ml. portions of water to extract traces of sulfuric acid. The

ether solution was then evaporated, and the residue taken up in ethyl alco-

hol and titrated with 0.01 N sodium hydroxide. No acid could be de-

tected. In a control experiment, in which 10 mg. of sodium oleate were

added to 0.2 gm. of amylose and hydrolyzed with sulfuric acid as described,

the oleic acid could be recovered quantitatively.End-Group Determination-A 0.2 gm. sample of the material was oxi-

dized with 0.37 M sodium periodate at 2” according to the method of Potter

and Hassid (29). After 25 hours, the excess periodate was removed by

the addition of ethylene glycol and the formic acid formed was estimated

by titrating with 0.01 N barium hydroxide. For the blank titration, 0.2

1 The nature of this material is now being investigated .

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S. NUSSENBAUM AND W. 2 . HASSID 677

gm. of synthetic amylopectin was added to the periodate, which had been

previously reduced with ethylene glycol. Because of the slight alkalinity

of the blank, 10 ml. of 0.01 N hydrochloric acid were added to both theblank and the sample, and a correction was made when the sample was

titrated with the barium hydroxide. The average number of glucose resi-

dues per end-group for the synthetic amylopectin, calculated on the basis

of 1 mole of formic acid produced per chain, was found to be 21. The

chain length of the parent amylose (corn) determined by this method was

490 glucose residues per end-group.

Determination of Molecular Weight-A 1 gm. sample of the product was

dispersed in formamide and acetylated at room temperature with acetic

anhydride in the presence of pyridine (30). The molecular weight was

determined on the acetylated product from osmotic pressure measure-

ments, with chloroform as a solvent. The method employed was the same

TABL E I

Osmotic Pressure Data for Acetylated Synthetic Amylopectin

Concentration, C

gm. per 1.

19.63

11.78

7.07

4.24

Rise in osmome ter Osmotic pressure, T;

cm gm. per s*. cm..

4.49 6.60 0.336

2.47 3.63 0.308

1.42 2.12 0.296

0.80 1.18 0.278

as that previously used for the determination of a variety of starch frac-

tions (30). The osmotic pressure was measured at several different con-

centrations (Table I).The intercept of the coordinate was determined by plotting r/C against

C, and the molecular weight was calculated by the van? Hoff equation.

From the intercept of the ordinate a value of r/C was obtained equal to

0.262 for the acetylated product. This value corresponds to a number

average molecular weight of 97,000 for the acetylated product. On this

basis, the molecular weight of the deacetylated synthetic polysaccharide

is 54,000, as compared to an average molecular weight of the parent amyl-

ose of about 100,000. This molecular weight is smaller than those obtained

for amylopectins of natural starches (30), but its magnitude is nevertheless

large enough to eliminate the possibility that the synthetic product con-

sis ts of unbranched fragments of amylose, such as would be produced by

the action of a-amylase.

Reducing Value of Synthetic Amylopectin-The reducing value of a 30

mg. sample of synthetic amylopectin was determined (24) and compared

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678 ENZYMATIC SYNTH ESIS OF AANLOPECTIN

with the reducing values of the parent amylose and of a few natural starch

fractions of known molecular weight. The soluble amylopectin samples

(30 mg.) were dissolved in 5 ml. of water. The insoluble amylose wasbrought into solution with 2 N sodium hydroxide and neutralized with acid.

The results are given in Table II.

The reducing value of the synthetic amylopectin appears to be of the

same order or magnitude as that of the parent amylose or of an amylopec-

tin subfraction of low molecular weight. The comparatively low reducing

value of the synthetic product indicates that the synthetic polysaccharide

cannot be degraded amylose.

Action of “Isophosphorylase” on Amylose-The experiments. of Bernfeld

TABLE II

Reducing Values and Molecular Weights of Synthetic Amylopectin and Related

Polysaccharides

Substance Mol. wt.0.01 N ~~cs fate*

po ysacchahde

Synthetic amylopectin. . . . . . . . . 54,0000.023

Corn amylopectin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3,000,000 0.003“ “ subfraction. . . . . . . . 290,000 0.026“ amy lose. . . . . . . . . . . .. . . . . . .. . . . . . . .. . . . . . . .. 128,000 0.037‘I amylodextr in.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6,700 0.190

* 2.4 ml. of 0.01 N ceric sulfate solution are equivalent to 1 mg . of ma ltose.

and Meutemedian (16-19) were repeated with an enzyme preparation from

potatoes, made according to their directions, with the following results.

A mixture consisting of amylose + phosphorylase + isophosphorylase+ inorganic phosphate (phosphate buffer), originally giving a blue color

with iodine, produced a purple color after 24 hours of incubation. When

the inorganic phosphate was eliminated from the reaction by substituting

Verona1 for phosphate buffer of the same pH, the reaction mixture stained

blue with iodine after incubation. However, it was observed that, when

the quantity of isophosphorylase was doubled or tripled, the presence of

either phosphate or phosphorylase was not necessary to convert amylose

into a purple-staining polysaccharide.One possible interpretation of these results is that the supposed “iso-

phosphorylase” preparation contains ar-amylase and that the combined

action of this enzyme with phosphorylase in the presence of inorganic phos-

phate is responsible for the conversion of the amylose into fragments that

stain purple with iodine. The phosphorylase also contributes to the degra-

dation of the molecule, owing to the small amount of cs-amylase impurity.

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S. NUSSENBAUM AND W. 2 . HASSID 679

However, phosphorylase alone at this phosphate concentration wi ll not

cause a change in iodine color when acting upon amylose. When the

amount of the isophosphorylase preparation is increased, the concentra-tion of a-amylase probably becomes suffic iently large to accomplish the

degradation of amylose alone without the aid of phosphorylase.

Reducing Value of Product of “Isophosphorylase” Action--Samples con-

taining 10 mg. of amylose, phosphorylase, phosphate buffer, and “iso-

phosphorylase” were mixed and allowed to digest for 24 hours. After

deproteinization by heating, filtering, and washing, it was found that the

sample possessed a reducing value which was considerably greater than

that of the samples in which the “isophosphorylase” or amylose was leftout. The increase amounted to 1.2 ml. of 0.01 N ceric sulfate titration

(equivalent to 0.5 mg. of maltose) per 10 mg. of amylose. This value is

of the order of magnitude that would be expected if the amylose were

degraded to chains of approximately 40 glucose.units in average length by

an ar-amylase.

Identification of Breakdown Products of Starch with “Isophosphorylase”-

A 0.5 gm. sample of amylose was converted with “isophosphorylase” prepa-

ration and the resulting material precipitated and dried. The materialpossessed the following properties. (1) It was soluble in water, but retro-

graded after remaining at room temperature for 3 or 4 days. (2) When

dissolved in hot water and cooled, it did not precipitate when methyl or

ethyl alcohol was added in 50 per cent concentration. Amounts of this

material formed from 50 mg. of amylose did not produce any cloudiness in

50 per cent alcohol. Less than 1 mg. of amylopectin or the synthetic

branched polysaccharide in the same volume wi ll produce a definite tur-

bidity. (3) Acetone was found to precipitate the polysaccharide. (4)

When the material was dialyzed against distilled water, most of the

purple-staining material was lost, indicating that the molecules are small.

The properties of this product are associated with those of amylose

degradation products, rather than with properties of branched polysac-

charide. On the basis of these experiments, the mechanism of Bernfeld

and Meutemedian for amylopectin formation involving “isophosphorylase”

cannot be accepted.

Note on the Properties of Synthetic Amylopectin

BY GERTY T. CORI AND BAR BAR A ILLINCWORTH

(From the Department of Biolo gica l Chemistry, Washington University

School of Medicin e, St. Louis, Missouri)

Enzymatic End-Group Determination-The method (31) is based on the

degradation of the polysaccharide by phosphorylase plus amylo-l ,6-glu-

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680 ENZYMATIC SYNTH ESIS OF AMYLOPECTIN

cosidase (debrancher). The latter enzyme is specific for hydrolytic scission

of the 1,6 linkages. The ratio of phosphorylated plus free glucose to free

glucose is determined. Control experiments were run simultaneously witha sample of the amylose from which the amylopectin had been synthesized

and with three branched polysaccharides (corn amylopectin, rabbit liver

glycogen, and dog liver glycogen), the end-groups of which had also been

determined by the periodate oxidation method (31). End-group values

pertaining to the dog liver glycogen were also available from methylation

experiments (32). The results are summarized in Table III.

Three separate experiments carried out with the synthetic amylopectin

TABLE I I I

Enzymatic End-Group Analysis of Synthetic Amylopectin and Related Polysaccharides

The digestion mixture contained 12 mg . of polysacch aride in 15 m l., 0.004M MgSOd, 0.001 M cysteine, 0.014 M KH zPO I, crystal l ine mus cle phosphorylase, andglucosidase as stated. Incubation was at pH 6.8 at 30”.

Experi-ment NC

5

6

7

Polysaccharide

Corn amylose

Q enzyme synthesized“ “ ‘I“ “ ““ I‘ “

Corn amylopectin‘I “

Rabbit l iver glycogen

Dog liver glycogen

-

ilTime oflcubation

hrs. ger cent1 131 612 661 672 821 842 1001 1031

100

Digested :nd-groul

per cent1.7

5.35.0

5.1

4.46.7

8.3

’ * i

_-

-

No. ofglucose

&dues pegend-group

19

20

20

23

15

12

-

‘u

_-

-

Glut-sidase

nits used

225022501900

3850385038503850

2250

3850

showed good agreement. An end-group of 5.1 per cent was obtained, cor-

responding to an average chain length of approximately 20 glucose units.

This value closely agrees with that of 21 units obtained by the periodate

oxidation method. The 1.7 per cent end-group found for amylose is prob-

ably due to amylopectin impurity. Amylopectin is rapidly attacked by

muscle phosphorylase, while the action of this enzyme on amylose is very

slow. This is shown by the fact that only 13 per cent of the polysaccharidewas digested. If this assumption is correct, the amylose would contain

approximately 5 per cent amylopectin.

Phosphorolysis of Synthetic Anzylopectin-The synthetic material was

subjected to the action of muscle phosphorylase (ninth crystallization) in

the presence of an excess of inorganic phosphate, and the lim it of phos-

phorolysis was compared with that of rabbit liver glycogen and of natural

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S. NUSSE NRAUM AND W. 2 . HASSfD 681

amylopectins. The per cent degradation was as follows: glycogen 37, corn

amylopectin 53, wheat amylopectin 44, synthetic amylopectin 34. The

outer branches of the synthetic polysaccharide are relatively short and arecomparable to those of glycogen.

Separate and Consecutive Degradation of Synthetic Polysaccharide with

Phosphorylase and Glucosidase-A sample weighing 37 mg. was submitted

to exhaustive phosphorylase action, as described in the preceding para-

graph. When the reaction had come to an end-point, 32 per cent of the

polysaccharide had been converted to glucose-l-phosphate. The remain-

ing polysaccharide (first limit dextrin) was isolated by several precipita-

tions with alcohol and submitted to glucosidase action in the absence ofphosphate. Glucose liberated was determined by a specific enzymatic

method (31). It was found that 3.7 per cent of the limit dextrin had

been converted to glucose, which indicates that one-half of the branch glu-

cose residues present in the original material has been spli t off. The glu-

cosidase was destroyed by heating and phosphorylase was again permitted

to act on the remaining polysaccharide, with the result that 36 per cent

of it was converted to glucose-l-phosphate. At this point, 58 per cent of

the original material had been degraded. In addition, an aliquot of the

first limit dextrin was submitted to an end-group determination; 7.7 per

cent end-groups were found. From the end-group concentration of the

original material (5.1 per cent) and the loss of 32 per cent by phosphorylase

action, the calculated value is 7.5 per cent. These results, which are very

similar to those recently obtained on glycogen and amylopectins (Larner

et al. (33)), are compatible only with a tree-l ike structure of the synthetic

polysaccharide, which therefore does not differ in this respect from natu-

rally occurring polysaccharides.

Activation of Crystalline Muscle Phosphorylase-The “primer’; action

(34) of synthetic amylopectin was compared with that of glycogen, natural

amylopectin, and amylose in reaction mixtures constituted as follows: 12

mM initial concentration of glucose-l-phosphate, 40 mg. per cent of polysac-

charide and muscle phosphorylase. The following ratios, of activities were

found: when no polysaccharide was added, 0; with glycogen, 100; with corn

amylopectin, 63; with synthetic amylopectin, 60; and with amylose, 7.

These figures vary somewhat with different polysaccharide-glucose-l-phos-

phate ratios. The conclusion can be drawn that the synthetic amylopectinpossesses almost the same activating power as natural amylopectin.

Molecular Size of Synthetic Polysaccharide-A 1 per cent solution of the

sample in water was examined in the Spinco analytical ultracentrifuge by

Dr. John F. Taylor. At 59,700 r.p.m., a single boundary was observed,

moving with a sedimentation constant (20’) of 3.1 S at the start and falling

to 2.8 S at the end. The spreading of the boundary indicates that the

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682 ENZYMATIC SYNTH ESIS OF AMYLOPECTIN

material is polydisperse, as has been observed from other polysaccharides.

The diffusion constant of this material has not been measured. If the

frictional ratio, flfo, were assumed to be similar to that reported for glyco-gen (1.6 to 1.9), the “mean” molecular weight would be from 31,000 to

41,000.

S U M M A R Y

Linear amylose can be transformed with Q enzyme from potatoes to a

branched polysaccharide possessing the chemical and biochemical proper-

ties of amylopectin. Its molecular weight, estimated from osmotic

pressure measurements, is 54,000, which is less than that of naturallyoccurring amylopectins.

The synthetic product is soluble in water, does not retrograde from solu-

tion, and stains purple-red with iodine. Chain length determination by

periodate oxidation shows an average of 21 glucose residues per end-group,

while with an enzymatic method a value of 20 was obtained.

The synthetic polysaccharide is degraded by @-amylase to the extent of

51 per cent and by crystalline muscle phosphorylase to the extent of 33

per cent. An analysis of structure by means of enzymatic degradationshows close resemblance to the structure of natural amylopectins. As a

“primer” substance for muscle phosphorylase, it is about as effect ive ‘as

naturally occurring corn amylopectin.

The claim that the synthetic amylopectin is in reali ty a combination of

amylose with fatty acids, simulating the properties of an amylopectin,

could not be confirmed. Neither could evidence be obtained for the

existence of ‘Lisophosphorylase” in potatoes, capable of synthesizing amy-

lopectin.

The authors express their thanks to Professor C. F. Cori for his reading

of the manuscript and for his many helpful suggestions.

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