J.J. Marshall

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.

Application of Enzymic Methods to the Structural Analysis of Polysaccharides: Part I

Publisher Summary This chapter discusses the application of enzymes for the structural analysis of polysaccharides. The classical techniques of structural analysis of polysaccharides—namely, fragmentation analysis (for example, by acid hydrolysis), methylation analysis, and periodate oxidation have been supplemented to an ever-increasing extent by enzymic methods. The use of enzymes in the analysis of polysaccharide structures has become so extensive that it is now appropriate to survey the progress made by the application of such methods. The chapter illustrates how the two types of method, enzymic and nonenzymic, may usefully complement each other. This result may be achieved in several ways. In its simplest form, it merely involves nonenzymic characterization of products of enzymic hydrolysis or—more commonly—characterization, by simple enzymic procedures of products of low molecular weight that have been obtained by nonenzymic methods. Enzymatic procedures can be used in conjunction with nonenzymic or other enzymic methods to obtain quantitative data relevant to the elucidation of polysaccharide structure.

Incomplete conversion of glycogen and starch by crystalline amyloglucosidase and its importance in the determination of amylaceous polymers

Amyloglucosidase [EC 3.2.1.3.] hydrolysesboth the 1 -~ 4and 1 ~ 6-bonds of starch and glycogen, and is reportedly capable of causing a quantitative conversion of these polymers into glucose. It has become an agent of choice for the specific and quantitative determination of amylaceous polymers. Thus it was reported from our Laboratory that Aspergillus niger amyloglucosidase could be used for this purpose [1 ]. Recently we detected an o~-amylase-like impurity in our preparation of A. niger enzyme and turned to the use of a crystalline preparation from Rhizopus niveus. The unexpected finding was then made that the amylase-free enzyme is unable in many instances to bring about a complete conversion of the starch components and glycogens into glucose. Adulteration of the glucamylase with a-amylase restores the conversion to a quantitative level.

Manipulation of the properties of enzymes by covalent attachment of carbohydrate

Covalent attachment of low molecular weight sugars and polysaccharides to enzymes alters greatly the properties of the enzymes in vitro and their behavior in vivo. Some carbohydrate—enzyme conjugates may have potential as therapeutic agents.

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].

Kinetic difference between hydrolyses of γ-cyclodextrin by human salivary and pancreatic α-amylases

Abstract γ-Cyclodextrin was found to be hydrolyzed by human salivary and pancreatic α-amylases (1,4-α- d -glucan glucanohydrolase, EC 3.2.1.1) at appreciable rates. The optimum pH for the enzyme reactions at 37°C in the presence of 0.1 M NaCl was at around pH 5, which was remarkably different from the optimum pH (pH 6.9) of the enzymes for starch. The K m value (2.9 mg/ml) of pancreatic α-amylase for γ-cyclodextrin was smaller than that (5.3 mg/ml) of salivary α-amylase at pH 5.3, while the V value of the former was 3.7-times larger than that of the latter. The hydrolyses of γ-cyclodextrin by both enzymes took place via the multiple attack mechanism. The degrees of multiple attack by salivary and pancreatic α-amylases for γ-cyclodextrin at pH 5.3 were 2.0 and 1.1, respectively. The distribution of maltodextrins produced by hydrolysis of γ-cyclodextrin by salivary α-amylase was suggested to be independent of the substrate concentration, while that produced by pancreatic α-amylase was presumably dependent on the substrate concentration.

Attachment of carbohydrate to enzymes increases their circulatory lifetimes

In previous reports [l-6] we have described the coupling of a number of enzymes to cyanogen bromide-activated dextran to produce soluble enzymedextran conjugates. Enzymes modified in this way have stability properties that are superior to those of the corresponding native enzymes. Thus conjugation of catalase, OLand (3-amylases, and trypsin with dextran decreased their susceptibility to heat inactivation [l-5] . Following conjugation, the stability of trypsin in the presence of protein denaturants such as urea and sodium dodecyl sulfate was markedly increased, and the enzyme became resistant to inhibition by naturally-occurring protease inhibitors [2,3] . Enzymedextran conjugates were also shown to be more resistant than the corresponding native enzymes to proteolytic inactivation, both autolytic [2,3] and by added exogenous proteases [5] . We have now investigated the behavior of two synthetic enzyme-dextran conjugates in experimental animals with a view to determining whether enzymes modified by attachment of carbohydrate are likely to have properties making them more useful than unmodified enzymes for the therapy of metabolic disorders that are responsive to administration of exogenous enzymes. In this report the effect of attachment of dextran on the in vivo behavior of Bacillus arnyloliquefaciens cu-amylase and bovine liver catalase is described. Our findings suggest a new approach to increasing the

A new serum α-amylase assay of high sensitivity

Abstract The use of chemically modified starch substrates for measurement of serum α-amylase activity is described. Action of α-amylase on such substrates, in the presence of excess fungal glucoamylase, results in the production of glucose in direct proportion to the amount of α-amylase present. The glucose produced is measured by a specific enzymic assay. Results obtained by using this new assay correlate well with activities determined by a conventional saccharogenic assay. The new method is of much higher sensitivity, and is less susceptible to interference, than are most other α-amylase assay methods.

Preparation and characterization of a dextran-amylase conjugate

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.

Characterization of the α-D-glucan from the plastids of cecropia peltata as a glycogen-type polysaccharide

Abstract A water-soluble polysaccharide extracted from the plastids of Cecropia peltata , a higher green plant, has been characterized by enzymic methods as a polysaccharide similar to phytoglycogen.