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An endo-1,3-β- d -glucanase from germinated rye was purified 58-fold by ion exchange chromatography on DEAE- and CM-cellulose, and by gel filtration on Bio Gel P-60. The enzyme had pH optima of 5.0 with laminarin and 5.7 with carboxymethyl pachyman and was stable up to 45°. Bovine serum albumin and certain chlorides increased the activity at low concentrations of buffer. With laminarin, the enzyme had a K m of 0.25 mg/ml and the MW was estimated to be 24 300. Isoelectric focusing indicated that the endo-1,3-/gb- d -glucanase was a basic protein. The properties of the enzyme are compared with those from other higher plants.

Incubation of a 15% solution of maltose with a brewer's yeast maltase preparation resulted in the formation of small amounts of oligosaccharides. These have been separated by charcoal-Cefite chromatography, and characterized by chemical and enzymic methods as isomaltose, panose and maltotriose. The same enzyme preparation also catalysed limited oligosaccharide synthesis from glucose. Possible mechanisms of saccharide synthesis are discussed and the properties of yeast maltase and of other maltases are compared.

Limit dextrinase was isolated from an extract of freshly germinated barley by precipitation with ammonium sulphate followed by continuous electrophoresis. Enzyme activity was assayed using pullulan as substrate. The purified enzyme can release α-(1→6)-linked maltosyl and maltotriosyl units from both oligosaccharide and polysaccharide substrates (e.g. amylopectin β-limit dextrin). It had no action on substrates containing α(1→6)-linked glucosyl residues, or on amylopectin, glycogen, or glycogen β-limit dextrin. The enzyme also synthesised branched oligosaccharides from maltose and various maltosaccharides; glucose was not a substrate for these condensation reactions.

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.

Extracts of malted barley have been shown by molecular sieve chromatography to contain at least five enzymes which hydrolyse β-glucosidic linkages. In order of decreasing molecular weight, these are two β-glucosidases, an endo-β-1,4-glucanase, an endo-barley-β-glucanase and an endo-β-1,3-glucanase. This last-named enzyme was purified about 60-fold and shown to be specific for substrates containing only β-1,3-glucosidic linkages. This enzyme was activated by calcium ions, and deactivated by EDTA, but was not affected by chloride ions or a sulphydryl reagent. The endo-β-1,4-glucanase, which was purified about 50-fold, hydrolysed glucans containing only β-1,4- or a mixture of β-1,3- and β-1,4-linkages. The two β-glucosidases were shown to be true glycosidases and not exo-β-glucanases. The changes which occur in the levels of the various enzymic activities during the malting process have been measured.

R-Enzyme and limit dextrinase of malted barley have been separated by continuous electrophoresis. The preparation of R-enzyme, which was free of α-amylase, hydrolysed the outermost inter-chain linkages of amylopectin and glycogen which had interior chain lengths exceeding five glucose residues. It had no action on glycogen with shorter interior chains, on pullulan, or on oligosaccharides containing α-1, 6-glucosidic linkages.

Maltosaccharides of degree of polymerisation (DP) 6-22 have been isolated in a chromatographically pure form, and their iodine staining properties studied under standard conditions. In the range DP 6 to DP 13, there is a logarithmic increase in the intensity of absorption of the iodine complexes; thereafter the relationship is almost linear. A plot of the reciprocals of wavelength of maximum absorption and DP indicates that there are three separate linear sections corresponding to DP 6–11, 12–18 and 18–22. Ammonium sulphate causes an increase in the intensity of absorption and wavelength of maximum absorption in all cases.

α-Amylase was prepared from an extract of malted rye flour by heat treatment to inactivate β-amylase and other contaminating carbohydrases, followed by continuous electrophoresis. This yielded two isoenzymes which differed considerably in physical properties, although their action patterns appeared to be similar. These findings have been compared with the results of studies on α-amylase isoenzymes from other sources.

The rate of hydrolysis of amylopectin by three different limit dextrinase preparations is only about 15–23% of that of amylopectin β-limit dextrin under similar conditions. On dilution of the enzymes there was no change in specificity. The factors controlling the specificity of the enzyme and the possible significance in vivo of the results are discussed.

Molecular-sieve chromatography of an extract from ungerminated rye indicated the presence of enzymes which hydrolysed cellobiose, laminaribiose and the β-glucans cellodextrin, laminarin and barley β-glucan. A purified endo -β-1,3-glucanase was prepared from the extract by ammonium sulphate fractionation and molecular-sieve chromatography on Biogel P60. The substrate specificity and some properties of the enzyme are reported and the in vivo role of the enzyme is discussed.

THE debranching enzymes are a most important group because, with the amylases and phosphorylases, they completely degrade starch and glycogen to the monosaccharide level. Some of the earlier preparations of debranching enzymes were not homogeneous and their use led to erroneous conclusions about their specificity. These views were later qualified by studies using more highly purified preparations. For example, some preparations of yeast isoamylase hydrolysed α-1,6-D-glucosidic linkages in both glycogen and oligosaccharide α-dextrins1, but the latter activity is now no longer ascribed to isoamylase2.

THE use of light-scattering methods for the determination of the molecular weight, size and shape of macromolecules has become of increased importance in recent years. Studies already made include those on cellulose acetates1, polystyrene and synthetic polymers2, and several proteins3,4. The influence of sodium chloride on the size and shape of a detergent–protein complex has recently been investigated using a light-scattering method5. The application of this method to the measurement of the molecular weight of several glycogens is reported in this communication.

Two patients who had severe unstable diabetes mellitus with frequent hypoglycemic episodes and who required soluble insulin for control are described. Both developed hepatomegaly. Quantitative and qualitative studies of liver glycogen and its related enzymes were performed. The liver glycogen content was high (8 per cent) but its molecular structure was virtually normal. Both the β-amylolysis limit and the phosphorylase activity appeared to be lower than normal but did not approach the levels associated with glycogen storage disorders. The values of the other enzymes tested were normal. Therefore it appears that the elevated liver glycogen levels seen in some patients with brittle diabetes mellitus are not the result of enzyme deficiency but are secondary to wide fluctuations in blood sugar and frequent doses of soluble insulin.

1. Enzyme preparations from 11 plant sources, from yeast and from the protozoan Tetrahymena pyriformis show nigerase activity, which, in most preparations, was 70-90% of that towards maltose. 2. These enzyme preparations also hydrolysed isomaltose, but there was a wide variation in relative maltase to isomaltase activity. 3. The maltase and nigerase activities of alfalfa and tomato preparations could not be differentiated by heat inactivation or inhibitor methods. However, with turanose used as a competitive inhibitor, evidence suggesting that maltose and nigerose are hydrolysed at different catalytically active sites in the alfalfa preparation was obtained. 4. It is probable that the alfalfa alpha-glucosidase exists as a mixture of isoenzymes.

Publisher Summary This chapter discusses the molecular structure of glycogens. Glycogens, from vertebrate, invertebrate, bacterial, and yeast cells, are multiply-branched molecules (molecular weight ∼10 7 ) consisting of chains of α-(1 → 4)-linked D-glucose residues. The chains that are arranged in a tree- or bush-like form, normally contain an average of about 12 D-glucose residues; 40-50% of these may be removed by β-amylase. The exterior portions of the chains are therefore longer than those in the interior of the molecules, where adjacent branch points are separated by only 3–4 D-glucose residues. Small variations in molecular structure are shown by glycogens from different biological sources. A small proportion of glycogens may be expected to have abnormal structures. In this connection, the isolation of maltulose (some 5 %) from α-amylolytic digest of glycogen of pregnant-rabbit liver would suggest that D-fructose may be an extremely rare but minor component of certain glycogens.