ACTION OF AMYLO- ,8GLUCOSIDASE AND PHOSPHORYLASE ON GLYCOGEN AND AMYLOPECTIN*

BY GERTY T. CORI AND JOSEPH LARNERt

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

(Received for publication, July 31, 1950)

It is shown in this paper that two enzymes are needed for the complete digestion of the branched polysaccharides, glycogen and amylopectin. The first is phosphorylase, which is specific for the a-l, 4 linkages between glu- cose residues. In the presence of inorganic phosphate, glucose units are split off from the outer chains as glucose-l-phosphate until the enzyme ap- proaches the a-l, 6 linkages at the branch points.’ After exhaustive phos- phorylase action a limit dextrin remains which, in the case of glycogen, is about 36 per cent smaller than the parent molecule (4). In this limit dextrin the glucose unit in the a-l, 6 linkage has been exposed and is split off by the second enzyme as free glucose, permitting phosphorylase to con- tinue its action, a process which repeats itself several times until the whole glycogen molecule is digested. The ratio of free to phosphorylated glu- cose proved to be characteristic for the type of branched polysaccharide undergoing digestion and forms the basis for an enzymatic end-group de- termination. It is also possible to explore the inner structure of poly- saccharides by separate and consecutive action of the two enzymes and this will be reported in a later paper. The name amylo-l ,6-glucosidase has been proposed for the new enzyme (5) ; since its action is hydrolytic, it seems unlikely that it plays a r81e in the formation of the a-l ,6 bond.

It was known for some time that crude muscle extracts were able to digest glycogen completely, a fact which can now be attributed to the presence of glucosidase. Swanson (6) found that muscle phosphorylase

* This work has been supported in part by a grant from the Corn Industries Re- search Foundation.

t Postdoctorate Fellow of the National Institutes of Health, United States Pub- lic Health Service 1948-50. This report is from a dissertation to be submitted by J Larner in partial fulfilment for the degree of Doctor of Philosophy in Biological Chemistry, Washington University.

1 That this linkage is between carbon atoms 1 and 6 has recently been definitely established by Montgomery et al. (1) who isolated and identified 6[a-n-glucopyrano- syl]-n-glucose (also referred to as brachiose or isomaltose) after exhaustive digestion of starch with taka-diastase. The same disaccharide has also been isolated from giycogen after acid hydrolysis (Wolfrom et al. (2)), the a-l,6 linkage being more re- sistant to acid hydrolysis than the a-l,4 linkage (3).

17

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18 AMYLO-1,6-GLUCOSIDASE

crystallized two to three times, when added in large amounts and allowed to act for 20 or more hours, digested glycogen up to 80 per cent, while crude potato phosphorylase preparations, under the same conditions, de- graded glycogen only 36 per cent. Hestrin (4), on the other hand, obtained muscle phosphorylase preparations which behaved as potato phos- phorylase. This discrepancy has now been explained by showing that mus- cle phosphorylase preparations after two to three crystallizations are still contaminated with a small amount of glucosidase and that seven to eight crystallizations are required to remove the last traces. Several treatments of dissolved crystalline phosphorylase with norit also free it of glucosidase.

EXPERIMENTAL

Preparation of Glucosidase-A rabbit muscle extract was prepared as described by Green and Cori (7). After removal from the extract, dia- lyzed for 3 hours, of an isoelectric precipitate at pH 5.7, the supernatant fluid was brought to pH 7.0 with solid KHCOs. Further treatment con- sisted in the removal of a-amylase by adsorption on starch and of inor- ganic and organic phosphate by dialysis for 18 hours against 1 per cent KC1 at 3”. Extracts so dialyzed contained from 4 to 20 y of inorganic phosphate per ml.

The amount of amyla7 present in muscle extract is large enough to interfere with the measud lnent of glucosidase activity, especially during long periods of incubation A simple procedure, which Somogyi (8) had used for the removal of amylase from plasma, was found to remove the enzyme completely from muscle extract. Corn-starch powder (U. S. P. grade) was stirred into the cold extracts (10 gm. per 100 ml.) for 10 minutes, removed by centrifugation, and the treatment repeated once or twice, fol- lowed by centrifugation plus filtration; no starch was dissolved. It is es- sential that each extract be tested for amylase activity after the starch treatment. This can be done in a qualitative way by adding a small amount of soluble starch to the muscle extract. The blue iodine color disappears gradually when amylase is present, while it remains unchanged for 4 hours or longer when the starch treatment has been effective. Gen- erally a quantitative test has been relied upon to demonstrate the absence of amylase. The dialyzed extract was diluted 1:5 and incubated at 30” with 0.8 mg. of glycogen per ml. Copper reduction was determined after deproteinization. For example, before starch treatment the reducing values (in terms of glucose), after 1, 2, 3, and 4 hours of incubation at 30”, corresponded to 8, 14, 20, and 27 per cent of the total reduction after acid hydrolysis of glycogen. Actually, even after 4 hours only a small amount of glucose was formed and the reduction was due mainly to the formation of dextrins and maltose. After starch treatment, no reducing substances

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G. T. CORI AND J. LARNER 19

or only traces appeared, even when undiluted extract was incubated with glycogen for 4 hours. The importance of this control test is emphasized by certain experiments to be described in a later section.

Another enzyme which would interfere but which was found to be ab- sent is phosphatase. Glucose-l-phosphate and Mg++ were added to dia- lyzed extracts diluted 1: 5 and incubated for 1 to 16 hours at 30” at pH 7. No free glucose or inorganic P was liberated. The glucose-l-phosphate was converted to glucose-g-phosphate, indicating the presence of phosphoglu- comutase.

Glucosidase activity was measured in a reaction mixture of 2.5 ml. con- taining 2 mg. of LD2 (glycogen, phosphorylase), 0.004 M Mg++, 0.014 M

phosphate buffer of pH 7, from 0.1 to 0.5 ml. of muscle extract, and an ex- cess of crystalline muscle phosphorylase. This made glucosidase the limit- ing enzyme in the degradation of LD and also served the purpose of mag- nifying the effect of glucosidase through the action of phosphorylase. The methods of deproteinization and determination of reducing power will be described in a separate section. The amount of glucose, both free and phosphorylated, set free during 10 minutes of incubation at 30” was ex- pressed as units of glucosidase activity, 1 y of glucose being equivalent to 1 unit. Good proportionality was found over a 5-fold range of dilution of the extract when the experiment was so arr Lged that no more than 40 per cent of the LD was used up during incu tion with the highest con- centration of glucosidase. When glucosidase .was allowed to act alone on LD in a phosphate-free medium, the reaction reached an end-point when about 3 per cent of the LD had been degraded. This system had obvious limitations for a satisfactory quantitative test and hence the device was adopted of adding phosphorylase.

The amount of glucosidase present in muscle extracts after various treat- ments is shown in Table I. The enzyme was somewhat purified as follows: It was found that the mother liquor of the first phosphorylase crys- tals (7) contained about 30 per cent of the extracted glucosidase.3 Frac- tionation of this material with (NH&SO* at pH 7 resulted in about a 4- fold increase in specific activity (units per mg. of protein) over that found in muscle extract. Glucosidase solutions lose activity, even at 5”, during

2 This abbreviation is used to designate the limit dextrin, LD, formed from gly- cogen by exhaustive action of phosphorylase. The preparation of LD consisted in incubating glycogen with inorganic phosphate and a sample of phosphorylase which had been freed of glycosidase by seven to eight recrystallizations. When the re- action had come to an end, the polysaccharide was isolated by precipitation with alcohol and the treatment with phosphorylase repeated a second time. A third

~~~~im-?n:~aus~~~rrr~Gl~~fk?-~~q~~~~~r~f”t~n~S~~~~~r~ 3 This explains why phosphorylase crystals were found to be contaminated with

glucosidase.

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20 AMYLO-1,6-GLUCOSIDASE

dialysis against water or weak salt solution (1 per cent KC1 at pH 7) and also during storage. In order to avoid losses the enzyme was kept frozen in small lots until ready for use. Addition of cysteine delays the loss of activity during storage. After 24 hours at 5” and pH 6.8, the aliquot 80 which no cysteine had been added had 940 units per ml.; the aliquot with cysteine (0.02 M) had 2140 units. After 48 hours the respective values were 960 and 1560 units.

Long dialyzed, amylase-free glucosidase preparations were used in ex- periments in which the action of this enzyme alone was to be tested. Al- though the glucosidase preparations contain phosphorylase, the latter en-

TABLE I

Activity of Amylo-1,6-glucosidase in Various Rabbit Muscle Preparations

The method of preparation of extracts, the composition of the reaction mixture, and the definition of units are given in the text. Incubation was for 20 minutes at 30”. The first five experiments were carried out with extracts obtained from dif- ferent rabbits.

Preparation

Dialyzed 3 hrs. “ 18 “ “ 18 “ 2 starch treatments “ 18 “ 2 “ “ “ 18 “ 2 “ “

Nother liquor of phosphorylase crystals 2 starch treatments O-25y0 saturated (NHh)?SOa 25-350j0 saturated (NH~)?SO~

Units per ml. enzyme solution

1900 1200

970 1000 1100

6800 3600 1800

Units per mg. protein

114 170

68 74 71

247 433 370

zyme was practically inactive, because only traces of inorganic phosphate were present.

IdentiJication of Reaction Product-In approaching the problem of the mechanism of action of the “debranching” enzyme, the following possi- bilit,ies were considered: (1) that the “debrancher” enabled phosphorylase to by-pass the a-l,6 linkages; (2) that one was dealing with a transglu- cosidase, which catalyzed an exchange of the a-l ,6 bonds for a-l ,4 bonds; (3) that there occurred a splitt,ing of the a-l,6 bonds by phosphorolysis; (4) a split,ting by hydrolysis.

If the first mechanism prevailed, one would expect to find brachiose or a branched oligosaccharide as reaction products of phosphorylase plus “de- brancher” action; if the second or third, one would find only glucose-l- phosphate; if the fourth, one would find glucose, maltose, or a straight chain oligosaccharide. In order to decide between these possibilities it be-

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G. T. CORI AND J. LARNER 21

came necessary to look for unphosphorylated sugars in digests of glycogen with phosphorylase plus “debrancher” and, if such were present, to iden- tify them. Of various methods paper chromatography seemed to offer the best analytical approach.

An unpublished method of D. French was used, in which the one-phase solvent system consists of butanol6, pyridine 4, water 3 (parts by volume). Good migration and separation of sugars were obtained when the enzyma- tic digests were prepared in the following way. In 15 ml. of reaction mix- ture were present 15 mg. of glycogen, 0.03 M phosphate of pH 7.2, cysteine 0.003 M, Mg++ 0.003 M, an excess of muscle phosphorylase, and about 6000 units of glucosidase. Glycogen digestion was complete in 20 minutes, whereupon 2 ml. of the mixed Fe and HgS04 reagent of West (see Steiner (9)) was added. After neutralization with solid BaC03, the filtrate was made acid with H$04, freed of Hg with H,S in the usual way, and aerated. The final filtrate was neutralized and evaporated at 35” under an air stream.4 When the volume was 1 to 2 ml., ethanol was added to 90 per cent concentration and the mixture kept overnight at 4”. Salts precipi- tated which were removed by filtration. The ethanol was removed by an air stream at 35”, and the volume reduced to about 0.5 ml. This was the material used for chromatography. Steiner (9) has shown that no loss of glucose or maltose occurs in this procedure.

Chromatography revealed the presence of one reducing sugar only, which was detected by spraying the paper with alkaline copper sulfate solution and heating 5 minutes at 100”. Color was developed by spraying with phosphomolybdate. This sugar was identified as glucose by its RF. That other sugars, had they been present, would have appeared in the chro- matogram was proved by adding maltose or brachiose, 1.5 mg. each, to the reaction mixture at the end of the incubation period. The RF values of the three sugars under the conditions of the experiment were glucose 0.38, maltose 0.22, brachiose 0.15. Control experiments showed that glucose-l- phosphate was not split by treatment with the West reagent.

The mechanism of action of the “debrancher” is outlined under Mech- anism 4 above, and consequently the name amylo-1,6-glucosidase seemed appropriateP The fact that only glucose and no larger fragments, such as maltose or a trisaccharide, were found indicates that hydrolysis of the a-l,6 bond takes place only after preceding phosphorylase action has re-

4 No inorganic or organic P was found in this filtrate. 6 Meyer and Bernfeld (10) described an enzyme in yeast juice which showed some

degradation of LD (starch, p-amylase) after 40 hours of incubation in the absence of phosphate. They proposed the name amyloglucosidase, but did not identify the products formed. It is therefore impossible to say whether the yeast enzyme is in any way related to the enzyme described here.

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22 AMYLO-1,6-GLUCOSIDASE

moved all glucose residues from one branch. The absence of maltose also proves that the enzyme preparations used did not contain amylase.

Quantitative Determination of Free and Phosphorylated Xugars-Owing to the presence of phosphoglucomutase and isomerase in the glucosidase preparations, the following equilibrium was established during incubation of glycogen with added phosphorylase and inorganic phosphate: glucose-l-P G glucose-6-P i+ fructose-6-P. The determination of phosphorylated sugar therefore had to include these three varieties. Free glucose, the product of glucosidase activity, had to be determined in a filtrate entirely free of phosphorylated sugars.

Aliquots of a digestion mixture similar to that described in the preced- ing section were deproteinized at zero time and after incubation. The sum of free glucose and phosphorylated sugars was determined in Schenk filtrates (2 ml. of reaction mixture + 2 ml. of 2.5 per cent HgClz in 0.5 N HCl), while glucose was determined in phosphate-free West filtrates (5 ml. of reaction mixture + 0.7 ml. of West’s reagent). In both procedures polysaccharides are removed by adsorption on the HgS precipitate. This is important because acid hydrolysis is used in one step of the determina- tion.

The Schenk filtrates (which contained about 0.3 N HCl after removal of Hg) were first heated for 5 minutes at 100” in order to split the (non- reducing) glucose-l-phosphate.6 After neutralization, Nelson’s (11) color- imetric procedure was used with Reagent 60 of Shaffer and Somogyi (12) and a heating time of 30 minutes. (KI was omitted, and copper sulfate and alkali solutions were mixed just before use.) This high alkalinity re- agent was chosen because glucose-6-phosphate (a sample of high purity) gave, on the basis of its phosphorus content, as much reduction per mole as glucose itself. It was therefore possible to determine the sum of glucose and hexose phosphates without having to apply corrections.

Free sugar in the West filtrates was determined by two independent methods, Nelson’s (11) copper reduction and a spectrophotometric en- zymatic method. In the spectrophotometric test, glucose was converted to glucose-6-phosphate with crystalline yeast hexokinase and adenosine- triphosphate. When this reaction had gone to completion, glucose-8phos- phate dehydrogenase and triphosphopyridine nucleotide were added and the reduction of the latter was followed at 340 rnp in the Beckman spec- trophotometer until it reached an end-point, about 30 minutes.’ Each

6 In incubated samples the increase in the copper reduction value after hydroly- sis (about 5 per cent) corresponded to the concentration of glucose-l-phosphate ex- pected from the mutase equilibrium.

7 Filtrates prepared according to West did not contain hexosemonophosphate, as shown by the absence of any reaction before the addition of adenosinetriphos- phate.

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G. T. CORI AND J. LARNER 23

microgram of glucose in a volume of 3 ml. in a 1 cm. absorption cell corre- sponded to a log IO/I reading of 0.0115. Recovery of glucose from stand- ard solutions containing 10 to 30 y was satisfactory and there was also good agreement (within f5 per cent) with the copper reduction values for glucose in the West filtrates. Since the enzymatic method as used is specific for glucose, the formation of this sugar by glucosidase action, originally detected in chromatograms, appears to be substantiated.

Specificity of Glucosidase-A model of the structure of an outer branch of LD (glycogen, phosphorylase) is shown in Fig. 1. The experimental findings on which this structure is based are as follows: Glucosidase alone, acting in a phosphate-free medium, liberates glucose from LD (glycogen, phosphorylase) but not from glycogen; hence the glucose unit in a-l,6

SIDE BRANCHES

PHOSPHORYLASE

FIG. 1. Structural model of a portion of a branched polysaccharide showing the sites of enzymatic action. LD corresponds to the limit dextrin formed by exhaus- tive action of phosphorylase. R refers to the reducing end.

linkage must first be exposed by phosphorylase action before glucosidase can hydrolyze this bond. This requires that all glucose units in a-l ,4 linkage be split off from one of the two outer branches originating from a branch point. This branch is designated as the side branch. That the other, or main, branch in the LD is still 5 to 6 glucose units long follows from the fact that /3-amylase liberates from LD (glycogen, phosphorylase) 1 maltose equivalent per outer branch (4). Actually, as shown in Fig. 1, the maltose units must all come from the main branch. One possible ex- planation for the unequal attack of phosphorylase on the two branches is the following. Fischer-Hirschfelder models of maltose and brachiose re- veal that the a-1,6 link is a much more mobile one than the (r-1,4 link, be- cause in the former case the link involves a carbon atom outside the py- ranose ring. It is conceivable that the greater mobility of the side branch permits its complete degradation by phosphorylase.

The specificity of glucosidase for the structure shown in Fig. 1 has been

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24 AMYLO-1,6-GLUCOSIDASE

tested in several ways. In one case a polysaccharide was used as substrate, which had been prepared from glycogen by non-exhaustive phosphorylase action; that is, the material corresponded to 30 per cent degradation in- stead of 36 per cent as required for the formation of ED. Only traces of glucose were liberated by the glucosidase, while LD (glycogen, phosphory- lase) yielded 2.7 per cent of free glucose.* In the case of glycogen incu- bated with glucosidase alone no reducing substances whatever were found in the West filtrate, even after 3 hours of incubation. Incomplete deg- radation of the side branch seems the most likely explanation for the lack of glucosidase action on the intermediate between glycogen and LD.

That the number of glucose units on the main branch also influences glucosidase activity seemed to be indicated by the slowness of its action on LD (amylopect.in, fi-amylase). One cannot be certain, however, that in this LD all glucose units on the side branch are exposed. P-Amylase removes 2 glucose residues (maltose) in one step; if, therefore, the side branch originally contained an even number of glucose units, the residue in a-l,6 linkage would remain covered by 1 glucose unit. In order to in- sure that this was not the case, LD (glycogen, phosphorylase) was treated with crystalline fl-amylase. In this manner an LD was obtained which differed from LD (glycogen, phosphorylase) merely by the length of t,he main branch, 1 to 2 glucose units instead of 5 to 6. While LD (glycogen, phosphorylase) was digested 18, 50, and 101 per cent after 10, 22, and 60 minutes of incubation with glucosidase plus phosphorylase, the LD with the shortened main branch was digested only 5 and 10 per cent after 60 and 120 minutes, respectively.

The reverse type of experiment was also carried out; namely, to make the main branch longer. LD (glycogen, phosphorylase) was incubated with phosphorylase and an amount of glucose-l-phosphate calculated to allow the addition of 1 glucose unit per outer branch at equilibrium of the reaction. In another experiment the added glucose units were 3.6 per outer branch; this should have yielded a material almost equal in size and properties to the original glycogen, if the glucose units had been randomly distributed between the t.wo branches. Both polysaccharides were iso- lated by several precipitations with ethanol and dried at room temperature. The iodine color of the (3.6) synthetic polysaccharide was reddish purple, with an absorption maximum at 525 rnp compared to 480 mp for glycogen. The synthetic material also differed in its solubility from glycogen, since it showed marked retrogradation when kept at 0”. These facts favor the assumption.that the glucose units were all added to the main branch, which

8 In these experiments with glucosidase alone, the incubation was continued until two consecutive periods showed no further change, indicating that the reaction had reached an end-point.

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G. T. CORI AND J. LARNER 25

would then be about 12 glucose units long. The iodine color and absorp- tion maximum are consistent with such a chain length (13).

The (1) and the (3.6) synthetic polysaccharides were submitted to glu- cosidase action; (1) gave 1.0 per cent of glucose, which is about one-third the amount obtained from the original LD, while (3.6) gave 0.25 per cent of glucose. Random distribution of 3.6 glucose units between the two branches should have yielded a synthetic polysaccharide completely re- sistant to the action of glucosidase. The experiments support the idea that lengthening of the main branch only occurred and that this inter- fered with the specificity requirements of the glucosidase, as did the shortening of the main branch.

The action of glucosidase on brachiose has also been tested. After in- cubation of 2 mg. for 6 hours with 3600 units of the enzyme some hydrolysis (about 25 per cent) could be demonstrated. The specificity requirements of the glucosidase are even less fulfilled in this case, since the a-1,6 link- age in the disaccharide is hydrolyzed at best at a small fraction of the rate at which it is hydrolyzed in the LD.

Enzymatic End-Group Determination-Chemical end-group determina- tions (by methylation or periodate oxidation) are based on the different reaction products obtained from the terminal glucose units with a free hydroxyl group on carbon 4, as compared to the other chain units with two, or at the branch points, with three connecting links. The numerical relationship between the number of end-groups and the number of branch points is as n to (n - 1) ; hence they may be regarded as practically equal in a large molecule. In t,he enzymatic method the glucose residue at a branch point is uniquely determined, since it is set free as glucose by ac- tion of glucosidase, while all other chain units appear in phosphorylated form. The ratio of free glucose to total glucose (free + phosphorylated) in a digest of a polysaccharide with phosphorylase plus glucosidase should therefore be a measure of the degree of branching, and inversely, of the average number of glucose units per branch.

In Table II are summarized enzymatic end-group determinations on various polysaccharides. All of these can be degraded completely by the two enzymes; incomplete digestion was deliberately chosen in some cases.g It may be seen that partial enzymatic degradation of glycogen and of its limit dextrin gave the same per cent end-group as complete degradation. This could be interpreted in two ways, either that. the struct’ure of the poly-

9 The per cent degradation was calculated from copper reduction values after complete acid hydrolysis (4 hours in 1 N HCl at 100”) of aliquots of the polysaccha- ride solutions used. These values obtained after acid hydrolysis generally agreed within 3 per cent with those calculated from the weight of the carefully dried poly- saccharides.

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26 A&n-Lo-l, 6-GLucoSIDASE

saccharides is very regular or that 1 polysaccharide molecule is degraded completely by the two enzymes before a new one is attacked.

The values for end-groups obtained by the enzymatic method showed a consistent deviation of about 20 per cent from the values obtained by

TABLE II End-Group Determination of Polysaccharides

The reaction mixtures consisted of 12 mg. of polysaccharide; MgSOa 0.004 M; inorganic phosphate 0.014 M, pH 7; muscle extract 3.0 ml. (approximately 3000 glucosidase units); phosphorylase crystal suspension diluted 1:20 with 0.003 M cysteine, pH 7, 5.0 ml.; final volume 15.0 ml. Time of incubation 15 to 60 minutes at 30’. Amylopectin samples were those previously analyzed by Potter and Hassid (14) with the periodate method. Periodate values on rabbit liver glycogen and corn amylopectin represent determinations made in this and Hassid’s laboratory, with substantial agreement.

Polysaccharide

Rabbit liver glycogen

“ muscle glycogen Limit dextrin of glycogen

(phosphorylase)

Sag0 amylopectin

Wheat amylopectin Sample A

‘I B Corn amylopectin

Potato amylopectin

per cent 42.5 90.5

102 102

64 100 100 96.6 97.4 76.5 89.6 90.5 92.6

85 97 98.3 88.5

Enzymatic

6.5 6.7 7.0 7.0 (6.8) 14.7 6.9 14.5

12.1 11.8 11.3 (11.7) 8.5 5.7 6.0 (5.9) 17 4.9 4.9 6.2 5.4 (5.4) 18.5 5.4 18.5 4.6 5.0 4.4 (4.7) 21.2 4.6 21.8

The figures in parentheses represent the average.

Periodate

5.55

4.55

4.35

3.84 3.7

18 18

22 22.5

23 20.

26 18.5 27 20

kviation

_-

-

)C? cent

periodate oxidation, the enzymatic values being higher in all cases. This suggests a systematic error in one of the two methods. The recovery of a known amount of polysaccharide in terms of free plus phosphorylated

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G. T. CORI AND J. LARNER 27

hexose after complete enzymatic digestion was within f2 per cent of the theory. This would not exclude a systematic error in the determination of the ratio of free glucose to total glucose, because free glucose, being only 5 to 7 per cent of total glucose, could be determined 20 per cent too high without affecting appreciably the per cent recovery of the enzymatically digested polysaccharide. It is therefore of imporbance that free glucose was determined by two independent methods, with a random error be- tween the two methods of f5 per cent.

Meyer and Rathgeb (15) have pointed out that esterification of formic acid may give low results in the end-group determination by periodate. This method is based on the titration of the formic acid liberated, and theoretically it should be sufficient to titrate to a methyl red end-point, as is done in the methods of Potter and Hassid (14) and Brown et al. (16). However, Jeanes and Wilham (17) have shown recently that, in order to get agreement between formic acid calculated from oxidant consumed and formic acid as determined by titration, one has to titrate to a phenol- phthalein end-point. There appears to be a buffering action in the critical pH region in incubated periodate-polysaccharide reaction mixtures (after removal of excess periodate with ethylene glycol) which is not found in solutions of pure formic acid. Further work is indicated in order to re- solve the discrepancy between the chemical and enzymatic method of end- group determination.

Glucosidase Action in Vivo-The liberation of free glucose from gly- cogen by glucosidase acting together with phosphorylase explains a hitherto puzzling finding made in this laboratory (18, 9). When rat or cat muscle in situ or isolated frog muscle was given three tetanic stimulations of 10 seconds duration each, a procedure leading to rapid breakdown of glycogen, a small amount of free glucose was shown to have been formed in the muscle (on an average 16, 23, and 13 mg. per 100 gm. in the three cases, respec- tively). West filtrates were prepared and differential analyses showed that only glucose and no maltose or non-fermentable reducing substances had been formed as the result of stimulation. In rat muscle in situ about 200 mg. of glycogen per 100 gm. are broken down during a tetanus of 15 seconds duration; hence probably somewhat more during three stimulations of 10 seconds duration. The amount of free glucose formed is of the order of magnitude one would expect if glucosidase participated in intact muscle in the breakdown of glycogen.

SUMMARY

1. The complete degradation of branched polysaccharides in animal tissues requires two enzymes, phosphorylase for the phosphorolysis of the a-l,4 linkages and a special type of glucosidase for the hydrolysis

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28 AMYLO-1 ,6-0~~00sI~AsE

of the a-l,6 linkages at the branch points. The product formed by glu- cosidase is glucose, the only free sugar which was detected in chromato- grams of suitably prepared digestion mixtures. The formation of glucose requires that the glucose unit in a-l ,6 linkage be exposed by preceding phosphorylase action. Thus, phosphorylase converts glycogen or amylo- pectin to a limit dextrin which yields glucose when acted upon by glucosi- dase alone, while native or incompletely degraded polysaccharides do not.

2. /3-Amylase liberates 1 equivalent of maltose per branch from the glycogen-phosphorylase limit dextrin; hence the main branch originating from a branch point is about 5 to 6 glucose units long, while the side branch consists of the exposed glucose unit on which glucosidase acts. Glucosi- dase has high specificity for this structure as shown in experiments in which the main branch was made longer or shorter. Glucosidase also acts very slowly on brachiose, the cw-1,6-linked disaccharide. The name am- ylo-1,6-glucosidase has been proposed for this enzyme and a method for the measurement of its activity has been described.

3. The ratio of free glucose to free + phosphorylated glucose in a digest of polysaccharide by the combined action of phosphorylase and glucosidase is a measure of the degree of branching. The values obtained with this enzymatic end-group determination on several branched polysaccharides were consistently 20 per cent higher than the values for end-group obtained by periodate oxidation. Possible reasons for this discrepancy are dis- cussed.

The authors are great.ly indebted to Dr. E. Montgomery for a sample of chromatographically pure brachiose, to Dr. A. K. Balls for a sample of crystalline P-amylase, and to Dr. W. 2. Hassid and Dr. R. W. Kerr for samples of several amylopectins. They also wish to thank Dr. C. F. Cori for valuable help and advice.

BIBLIOGRAPHY

1. Montgomery, E. hl., Weakley, F. B., and Hilbert, G. E., J. Am. Chem. Sot., 71, 1692 (1949).

2. Wolfrom, ill. L., and O’Neill, A. N., .I. Am. Chem. Sot., 71, 3857 (1949). 3. Swanson, M. A., and Cori, C. F., J. Biol. Chem., 172, 797 (1948). 4. Hestrin, S., J. Biol. Chem., 179, 943 (1949). 5. Cori, G. T., and Larner, J., Federation Proc., 9, 163 (1950). 6. Swanson, M. A., J. Biol. Chem., 172, 805 (1948). 7. Green, A. A., and Cori, G. T., J. Biol. Chem., 161.21 (1943). 8. Somogyi, hl., J. Biol. Chem., 126, 399 (1938). 9. Steiner, A., Proc. Sot. Exp. Biol. and Med., 32,968 (1935).

10. Meyer, K. II., and Bernfeld, P., Helv. chim. acta, 26, 399 (1942).

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G. T. CORI AND J. LARNER 29

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Gerty T. Cori and Joseph LarnerGLYCOGEN AND AMYLOPECTIN

AND PHOSPHORYLASE ON ACTION OF AMYLO-1,6-GLUCOSIDASE

1951, 188:17-29.J. Biol. Chem. 

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