ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 161, 234238 (1974)

Multiple Branching in Glycogen and Amylopectin’

J. J. MARSHALL AND W. J. WHELAN

Department of Biochemietrg, University of Miami School of Medicine, Miami, Florida S316R

Received November 21, 1973

The fl-amylase limit dextrins of glycogen and amylopectin are completely de- branched by joint action of isoamylase and pullulanase. Action of isos.mylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half t,he total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the p-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains:B-chains is markedly higher in amylo- pectins (152.6:1) than in glycogens (0.6-1.2:1). Glycogen typically contains A- chains and B-chains in approximately equal numbers; amylopectin typically con- tains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.

Enzymic methods for the structural ex- amination of glycogen and amylopectin have enabled the strucutres of these mole- cules to be defined with considerable preci- sion, much beyond that for similar types of polysaccharide for which the biochemist does not yet have comparable enzyme probes (1, 2). It has to be faced, however, that the full details of the fine structures are far from complete. While these are seemingly simple polysaccharides, containing a single mono- meric unit and only two linkages, it is the presence of the minor (branch) linkage that gives rise to an infinite variety of possible structures, which have to be narrowed down t’o what, in any event, can only be an average description, considering that there must be wide variations in the relative arrangements of the two linkages both inter- and intramolecularly.

1 This investigation was supported by National Science Foundation Grant GB 27491.

In the attempt more precisely to describe the structures, we defined three types of unit 1,4-cy-glucan chain, A,B, and C,2 mutually interconnected through the 1,6- branch linkages. There is only one C-chain per molecule. An important parameter is the ratio of A:B chains because this describes the degree of multiple branching. Several enzymic methods have been described for the measurement of this ratio (3-5). All the methods are time-consuming, either in requiring chromatographic separations of debranched products, and/or a knowledge of the average unit chain lengths of the polysaccharides.

2 A-chains are those which are linked to the rest of the macromolecule through a 1,6-oc-gluco- sidic linkage involving the potential reducing end residue; B-chains are also joined in this way but in addition carry substituent chains joined to primary hydroxyl groups; the C-chain has the only free reducing group in the molecule (24).

234

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MULTIPLE BRANCHING 1N GLYCOGEN AND AMYLOPKCTIN 235

We have rtrccntly discovered (6) in a species of Cytophaga a debranching cnzymc, isoamylase, which hydrolyzes the interchain linkages in glycogen, amylopectin, and certain degradation products of these poly- saccharides. The specificity of this enzyme, which is such that it can readily hydrolyze 1 ,G-bonds involving A-chains of three glu- cose units in length, but not t\vo-unit A-chains (6, 7), has made it useful in inves- tigations of the fine structures of glycogen and amylopectin. Thus we have been able to propose (8) a modification of the ramified st>ructure suggested by Meyer and cowork- ers (9, 10) for these polysaccharides, which givcls a more accurate description of the arrangcmcnt of the unit chains in the poly- saccharides.

This publication shows how the specificity of Cytophaga isoamylase may be used to advantage to obtain a rapid and accurate quantitative measurement of the ratio of A:B chains, and thcrcfore of t’hc extent of multiple branching in samples of glycogen and amylopcctin. The results show that t’here arc marked differences in the ratio b&wren thcsc two groups of polysaccharides. A prc>liminary account of this work has been published (7).

MATERIALS AND METHODS

:UuleriaZs. CzJlophaga isoamylase (glycogen G- glucanohydrolase, EC 3.2.1 .F8) was partly puri- fied using DEAE-cellulose as previously described (ll), followed by chromatography on a column of Biogel P-100 (J. J. Marshall, unpublished work). The sample used had a specific activity of 7.0 nkatjmg protein3 and was free from contaminating carbohydrate. Pullulanase (pullulan O-glucano- hydrolase, EC 3.2.1.41) from Aerobacter aerogenes was prepared and crystallized as described by Mercier et al. (12), and had a specific activity of 0.38 rkat/mg protein. Crystalline sweet-potato p- amylase (l,i-a-glucan malt,ohydrolase, EC 3.2.1.2) was prepared by a modification (J. J. Marshall and W. J. Whelan, unpublished work) of the method of Nakayama and Amagase (13), fol- lowed by chromatography on DEAE-Sephadex A-50 to remove contaminating or-glucosidase ac- tivity (14), and had a specific activity of approxi- mat,ely 33.3 wkat/mg protein.

Waxy-maize starch was prepared as by Schoch

3 Bbbreviation: kat, kat,al.

(15); waxy-sorghum starch was from Professor D. J. Manners. Other amylopectin samples were prepared by butanol fractionation (16) of solutions of the corresponding starches. Rabbit-liver glyco- gen was prepared as by Mordoh et al. (17) and phytoglycogen as by Schoch (15). Shellfish glyco- gen was purchased from Mann Research Labora- tories, and other glycogen samples were from Professor D. J. Manners.

Afefhods. Glucose was determined by the gill- case oxidase method of Lloyd and Whelan (18). Concentrations of polysaccharide solutions were determined by enzymic hydrolysis to glucose using a mixture of Aspergilllts rliger gluroamylase and oc-amylase (19). Measurements of reducing power were made with a copper reagent. (20), calibrated against glucose.

RESULTS

Preparation of P-alnylase limit dextrins of polysaccharides. P-Amylase limit dextrins of glycogens and amylopectins \vcre prepared by exhaustive treatment of solutions of the polysaccharides (20-30 mg/ml in 100 mM acetate buffer. pH 4.8) with fl-amylase (1.67 pkat/ml) at, 37°C \vith dialysis to remove maltose. Since the P-amylase used showed no instability under the conditions employed, the stabilizing agents, reduced glutathionc and bovine serum albumin, added previously (21, 22), were omitted. After digestion, the solutions ncbre boiled for 15 min to inactivate fl-amylasc, centri- fuged, dialysed, and the P-amylase limit dextrin solutions so obtained (concentra- tions generally within the range lo-20 mg,’ ml) were stored frozen until required.

Determination of yeduciug power liberated by action of debranching enzymes on /3-amylase limit dextrins. The amounts of reducing sugars liberated in 24 hr by the actions of (a) isoamylase and (b) isoamylase plus pullulanase on these dextrins were deter- mined in digests containing substrate solu- tion (50 ~1, prepared as above), buffer (acetate, pH 5.5, final concn 40 mM) and debranching enzyme(s) in a total volume of 0.5 ml. Isoamylase was used at a final con-

centration of 1.7 nkat/ml in both cases. In the case where both debranching enzymes were used the pullulanase concentration was 10 nkat/ml, and was added after 12 hr pre- incubation of the digests with isoamylase

236 MARSHALL AND WHELAN

alone. This was necessary since, when both enzymes were present thoughout the du- ration of incubation, complete debranch- ing did not take place. This phenomenon, which we attribute to inhibition of one de- branching enzyme by the other (cf. Ref. 23), will be discussed in detail elsewhere. When the degradation was carried out using the two debranching enzymes in the manner described, complete debranching took place, as evidenced by the quantitative conversion of the products into maltose by action of P-amylase. Appropriate control digests were also included.

Calculation of relative numbers of A-chains and B-chains in the polysaccharides. The method we have devised for determination of the degree of multiple branching takes advantage of the specificities of isoamylase and pullulanase to obtain a measure of the relative numbers of A-chains and B-chains in the P-amylase limit dextrins and thus, since /%amylolysis of the parent polysaccha- rides does not alter these values, in the unde- graded polysaccharides themselves. Action of isoamylase + pullulanase results in com- plete debranching so that measurement of the reducing power liberated gives the total number of chains (A + B) in the molecule. Measurement of reducing power after dc- branching with isoamylase, on the other hand, gives the number of B-chains, plus those A-chains which are debranched by isoamylase, namely those which are 3 units long. Since in the original polysaccharides, odd-numbered and even-numbered A-chains must, statistically, be present in equal numbers, there are equal numbers of 2-unit and 3-unit A-chains in the &dextrins, so that half the A-chains are debranched by isoamylase. Thus isoamylase action gives a measure of all the B-chains and half the A-chains (0.5 A f B). By difference from the total debranching value (A + B), half the number of A-chains is found and thence, by simple arithmetic, the relative numbers of A-chains and B-chains are obtained. It may be noted that these values could also be found by using the p-amylase-treated phosphorylase limit dextrins (4 ,&dextrins) described elsewhere (8, 21), in which all the A-chains are 2 units in length. In this case,

debranching with isoamylase would give a measure of the P-chains alone, and the difference from the total debranching value (A + B) would give the number of A-chains. However, since the fl-amylase limit dextrins are much easier to prepare than the +,a- dextrins, we have used them in the present work.

Table I shows the results of determina- tions of the ratios of A- and B-chains in several samples of glycogen and amylo- pectin.

DISCUSSION

We see from Table I that there is a sig- nificant variation in the ratio of A-chains:

TABLE I

DETERMINATION OF RATIOS OF A- AND B-CHAINS IN L%M~LES OF GLYCOGEN AND AMYLOPECTIN

Sample Reducing power

liberated by iso-

amylase 6s pg

glucose)

Reducing Number power of

liberated A-chains by iso- per

amylase B-chain0

‘rat2F (as rg

glucose)

Glycogens

Ascaris lumbri- coides

Shellfish Phytoglycogen Trichomonas

foetus Rabbit liver Human muscle Cat liver

Amylopectins

Wheat Rice Maize Potato Sweet potato Sweet corn Waxy maize Waxy sorghum

53.5 66.2 0.6

85.7 110.0 0.8 47.9 62.4 0.9 52.9 70.5 1.0

38.4 51.7 1.1 97.7 131.8 1.1 47.3 65.0 1.2

73.8 105.2 1.5 63.0 89.5 1.5 69.4 101.0 1.7 46.6 70.0 2.0 32.8 49.2 2.0 62.4 95.8 2.3 56.7 88.9 2.6 18.9 29.6 2.6

a A:B-chain ratio = 2(reducing power liberated by isoamylase + pullulanase - reducing power liberated by isoamylase)/[2(reducing power liber- ated by isoamylase) - reducing power liberated by isoamylase + pullulanase]: 1.

MULTIPLE BRANCHING IN GLYCOGEN AND AMYLOPECTIN 237

B-chains within the glycogens and within the amylopectins. Furthermore, there is a clear difference in this ratio between the t’wo types of polysaccharidc. The A:B-chain ratio, and thus the extent of multiple branching, is significantly higher in amylo- pectins than in glycogens. The average value of A:B for the glycogens is around unity, and models drawn to depict both glycogen and amylopectin have usually assumed a ratio of unity. However, the A:B-chain ratio in amylopcctins is, on average, closer to 2: 1. We may note in passing that when earlier, more limited, examinations of the A:B-chain ratios in amvlopectins and glycogens n-we made, similar results were obtained. Larner et al. (3) examined two glycogens and two amylo- pectins using the muscle glycogen-debranch- ing enzyme system, and the results, inter- preted in terms of our A- and B-chain ter- minology,2 agree with our present conclu- sions. The waxy-maize amylopectin exam- ined by Peat et al. (a;), using the plant’ starch-dcbranching enzyme, R-enzyme, may now be seen to have had an A:B-chain ratio closer to 2: 1 than to unity. Our more recent work, on the behavior of isoamylase towards glycogen and amylopectin 4, ,&dextrins re- vealcd corresponding differences between the polysaccharides (8). The only contrary note is struck in two publications by Man- ners. Results that showed that the ratio A:B was always less than unity for both glycogens and amylopectins, and could be as low as 1: 2.9 for each type of polysaccha- ride (4), were based on an incorrect model of the phosphorylase limit dextrin. Later results by Bathgate and Manners (5) were used to conclude that there was no signifi- cant difference between the A:B-chain ratio in glycogens and amylopectins. Pullu- lanase was used to debranch the p-dextrins, and the maltose and maltotriose from the A-chains \vas collected. The untested as- sumption was made that all the A-chains had been liberated by the enzyme a&on. We are alvare that pullulanase brings about total debranching of amylopectin fi-dextrin (26). Glycogen fl-dextrin, by contrast, is far from being compleMy debranched by

pullulanase (26). We know of no evidence that suggests t’hat the obvious inability of the enzyme to at’tack certain chains in the glycogrn @-dextrin is confined to the B-chains.

When presenting evidence on the be- havior of glycogen +,p-dextrins that was incompatible with the Meyer-Bernfeld model (S), n-e offered an alternative struc- t,ure in which the A:B-chain ratio is unity. The present results rcinforcc any structure for glycogen in which the ratio is unity, but require a revision of the model for amylopectin. Such a revision would come by adding A-chains to bring the ratio to 2 : 1. We begin to see in t’his distinction between amylopectin and glycogen a basis on which to attempt an explanation of the well- known differences in physical properties, particularly viscosity, between t)hc two polysaccharides; a more elongated structure has been proposed for amylopectin (27-30).

These results also permit us to comment on Erlandcr’s hypothesis that amylopectin is formed from glycogcn by dcbranching (31-33). The hypothesis has previously been criticized on negative grounds, such as the failure to detect a debranching PIN-

zymc which will carry out the transforma- tion, and the limited occurrence of glycogen in plants (34). Attent’ion is drawn to Table I in which sweet-corn amylopectin is seen to have an A:B-chain ratio of 2.3: 1 and sweet-corn glycogen (phytoglycogen) one of 0.9:1. Conversion of the latter into the former requires an increase in t)he ratio ,4:B. Erlander proposes that the conversion occurs by debranching. It is impossible to increase the proportion of A-chains by debranching. This could only be dont: by branching.

The procedure we have described for de- termining A:B-chain ratios in glycogen and amylopectin is particularly simple, requiring three readily available enzymes, and a simple type of measurement, that of rcduc- ing power. It is not even necessary t’o know the concentration of polysaccharide. We draw attention to a possible pitfall inherent in any method of determining the A:B- chain ratio based on the use of a limit dextrin produced by 311 exo-enzyme. All

235 MARSHALL AND WHELAN

reported methods except one (35) use such dextrins. The pitfall is the “buried chain,” a term used by French (36) to describe a chain whose nonreducing terminus, the point of attack by the cxo-enzyme, lies in- side the macromolecule, and is inaccessible to the exo-enzyme. The result of such failure to attack and attenuate an A-chain would be to give an A:B-chain ratio that is too low. WC have no information to offer on whether there are buried A-chains and, therefore, on whether this phenomenon will constitute a serious source of error.

ACKNOWLEDGMENT

We thank Professor D. J. Manners for the gift of several polysaccharide samples.

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