Eric E. Smith

A glycogen-debranching enzyme from Cytophaga

Potato R-enzyme [1 ] and bacterial pullulanase [2, 3] hydrolyse the a-1,6-linkages in pullulan and a-limit dextrins and also cleave the a-1,6-branch linkages in amylopectin [4]. The availability of highly purified preparations of pullulanase from Aerobacter aerogenes [5] has made this enzyme invaluable in the analysis of the fine structure of amylopectin [6]. Pullulanase, however, is of limited use in the analysis of glycogen structure because, although it is able to hydrolyse some branch linkages in degraded glycogen, it has little or no action on the undegraded macromolecule [7]. Yeast isoamylase [8] hydrolyses a limited proportion of the interchain linkages of amylopectin and glycogen but, unlike pullulanase, does not act on the 1,6-linkages of pullulan. Recently, an extracellular isoamylase from a new strain o f Pseudomonas was reported to hydrolyse almost all the branch linkages of amylopectin and glycogen [9, 10]. The importance of this type of enzyme for the analysis of glycogen structure prompts us to report the discovery of an isoamylase in a species of Cytophaga. Studies of the partially purified enzyme indicate that its specificity of action is similar to that of the Pseudomonas enzyme and that it has the ability to hydrolyse the branch linkages of amylopectin and glycogen with the complete dismemberment of the branched macromolecules.

The mechanism of Q-enzyme action and its influence on the structure of amylopectin

Abstract Q-Enzyme is responsible for the synthesis of the 1,6-branch linkages in amylopectin. Its action on a model amylodextrin containing a single branch linkage has been studied. It is concluded that the enzymic process whereby the branch linkages of amylopectin are synthesized is a random action of the branching enzyme on a complex—possibly a double helix—formed between two 1,4-α-glucan chains. This action pattern predicts a novel arrangement of the units chains in amylopectin.

Enzymatic determination of the unit chain length of glycogen and related polysaccharides

Enzymic methods for the determination of the average unit chain length (CL) of branched amylaceous polysaccharides are superior to chemical methods on account of the precision, rapidity and specificity of the former. These methods employ one, two or three enzymes, i.e. ol-amylase [I] , fl-amylase + pullulanase [2] and /.I-amylase + the amylo-1,6-glucosidase/oligo1,4-l ,4-transglucosylase complex of rabbit muscle or yeast [3], the last method being an adaptation of an earlier method [4] employing phosphorylase in place of /I-amylase. The a-amylase method is not absolute since it depends on a calibration with polysaccharides whose r% have been determined by other methods. The second and third methods are absolute and rely on the release and specific assay of the glucose units at the reducing termini of the unit chains. We now describe a method, based on the use of a single, commercially-available enzyme, which is both absolute and rapid. The enzyme is Cyrophuga isoamylase [S] which completely debranches glycogen and amylopectin. The assay is of the copper-reducing power of the reducing ends set free. The amount of polysacharide required for an assay may be as little as 200 I-(g and there is the additional advantage that, unlike the other enzymic methods, the unit chains are not depolymerized. Therefore the distribution in length of the unit chains can be examined by gel filtration [6].

Purification of dextran-binding protein from cariogenic Streptococcus mutans

Abstract An extracellular protein produced by Streptococcus mutans was purified to electrophoretic homogeneity by affinity chromatography on Sephadex G50 followed by gel filtration. The protein is devoid of both dextransucrase and dextranase activity but binds dextran and therefore probably is implicated in the adherence of S. mutans cells to the host tooth surface. The presence of the dextran-binding protein may be a determinant of the pathogenicity of such cariogenic micro-organisms.

On the specificity of starch debranching enzymes

An enzyme preparation from broad beans that hydrolyses the I + 6-branch linkages of amylopectin, amylopectin P-limit dextrin and amylopectin a-limit dextrins was discovered by Hobson, Whelan and Peat [l] and termed R-enzyme. Subsequently MacWilliam and Harris [2] described the fractionation of bean and malted barley extracts on alumina such that separate 1 + 6-bond hydrolases were found. One debranched amylopectin and its Pdextrin and not oligosaccharide a-limit dextrin. The second had the reverse specificity. These separated activities were given the respective names R-enzyme and limit dextrinase. Manners and coworkers [3,4] have subsequently confirmed the MacWilliam and Harris finding, with the same and with different plant extracts. In our own work, when further purifying potato R-enzyme, we have never observed a separation of the activities. Moreover, as we shall report here, a bacterial R-enzyme, pullulanase, has all the activities of R-enzyme, even when purified to homogeneity. Our inability to correlate our own results with those of MacWilliam and Harris [2] and Manners and coworkers [3,4] has perhaps been overcome following a recent report by Manners, Marshall and Yellowlees [S] , where it is stated that amylopectin P-limit dextrin is a substrate for “limit dextrinase”. This is contrary to the report by MacWilliam and Harris [2] but now permits an explanation of what limit dextrinase might be, since under its new definition, it has all the activities originally ascribed to Renzyme [ 11, save that of attacking amylopectin. We report here that when R-enzyme and pullulanase are diluted, the activity towards amylopectin selectively disappears,

Temperature-dependence of the action of Q-enzyme and the nature of the substrate for Q-enzyme

The 1,6-branch linkages that interlink the unit chains of amylopectin are formed by the action of Q-enzyme (EC 2.4.1.18) [l]. The most extensively studied variety of this enzyme is from potato, also the subject of this report. The enzyme acts by transglycosylation and we have demonstrated that this event can occur by inter-chain transfer of a chain fragment from a donor chain to an acceptor chain, a 1 ,Cbond being broken in the donor chain and a 1,6-bond being formed between the transferred fragment and the acceptor chain [2] . Intra-chain transfer, in which the donated portion is added to the residue of the chain from which the fragment was severed, has not been excluded. There must obviously be minima to the lengths of the donor and acceptor chains. One of us reported some 20 years ago that when phosphorylase was allowed to elongate short maltodextrin chains in presence of Q-enzyme, action of the latter was not seen until the chains had reached an average chainlength (CL) of about 40 [3]. The experiment did not permit a decision to be made whether the minimum length so observed was that of the donor, or the acceptor chain, or both. This observation has remained a paradox. An extended chain of 40 1 ,Clinked qlucose units would be 170A in length. This is considerably longer than the maximum possible size of the combining site of Q-enzyme (mol. wt about 85 000) [4]. With the demonstration that Q-enzyme acts by inter-chain transfer (see above) we have come to realize the possible significance of the otherwise puzzling observation. Inter-chain transfer could occur

The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R.

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran--cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.

Evidence for the periplasmic location of glycogen in Saccharomyces.

Abstract Treatment of yeast cells with hot alkali fails to solubilize a significant amount of glycogen. The insoluble glycogen is readily hydrolyzed by insolubilized α-amylase indicating that the apparent insolubility of this glycogen fraction does not result from its physical entrapment within an insoluble glucan membrane. The alkali-insoluble glycogen fraction of glutaraldehyde treated-cells is rapidly degraded by a mixture of snail gut enzymes during the formation of spheroplasts but the alkali-soluble glycogen fraction is unaffected. These results indicate that a major fraction of yeast glycogen is located outside the cellular membrane.

The substrate specificity of neutral α-glucosidase from rabbit muscle

Rabbit muscle contains different amounts of two α-glucosidase fractions possessing neutral pH optima. The fractions are separable by ammonium sulfate precipitation and have been partly purified and compared. They exhibit identical substrate specificities, inhibitor kinetics, and pH optima, and cannot be distinguished by these parameters from a 1000-fold purified preparation of the major neutral α-glucosidase. It is concluded that the two-cell homogenate fractions represent the same activity associated with different cell components. The 1000-fold purified glucosidase has a pH optimum of 7.4 (maltose), is noncompetitively inhibited by divalent cations (Ki 1 m M Zn2+, 0.3m M Cd2+), competitively inhibited by Tris (Ki, 4m M ), and slightly inhibited by polyols. The Km for action on maltose is 2.1 m M , on maltotriose 1.2 m M , and on panose (62-α-glucosylmaltose) and isomaltose, 16.7 m M and the relative V values are 1.0, 0.6 and 0.4 (panose and isomaltose), respectively. As evidenced by its action on maltodextrins and panose, neutral α-glucosidase possesses constitutive 1,4- and 1,6-α-glucanhydrolase activities, but it has little action on glycogen. The neutral α-glucosidase, therefore, is an oligosaccharase and probably acts on glycogen in conjunction with α-amylase. An ancillary role in the control of glycogen structure is proposed for the combined action of the two hydrolytic enzymes

On the affinity of rabbit-muscle glycogen phosphorylase for highly branched macromolecular subtrates

Abstract The rapid actions of mammalian muscle phosphorylases on glycogen and amylopectin may not result from their high affinity for the polysaccharide unit chains but from the high concentration of chain ends at the polysaccharide surface. When set free by the debranching action of pullulanase the linear unit chains of amylopectin are acted on at a low rate by the mammalian enzymes in contrast to the rapid rate of reaction catalyzed by potato phosphorylase. These findings suggest that the conformation of the active site of the mammalian phosphorylases compensates for the weak binding of individual chain ends by allowing the enzyme to act, without hindrance, on the densely packed polysaccharide chain ends at a near-maximum velocity.