8-Glucosidases electrophoresis

SEC purified -D-g ucosidase. This enzyme grade was prepared by applying diafiltered SP188 to the Sephacryl S-200 column. The column was operated at a flow rate of 15 mL/min in 10 mM phosphate buffer pH 6.5 with 100 mM NaQ. -D-glucosidase activity was found in an early eluting peak which proved to be approximately 92% -D-glucosidase by SDS-polyacrylamide gel electrophoresis (PAGE)(data not shown). 

Electrophoresis, of starch, I, 251 Emicymarin, I, 148, 156 Emulsin. See also j9-Glucosidase. Emulsins, V, 60-66... 

A search for lysosomal hydrolases and related enzymes has been made in haemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for a-D-mannosidase, neutral o-D-glucosidase, and -D-2-acetamido-2-deoxyhexosidase. a-D-Mannosidase activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5-6.0. Electrophoresis on cellulose acetate showed three bands. Haemolysates from four patients with mannosidosis were not deficient in a-D-mannosidase. Curves of pH activity and electrophoretic patterns were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell a-D-mannosidase differs from the lysosomal acid a-D-mannosidase. 

Storage of human saliva, serum, and duodenal secretion transformed the amylase fractions on cellulose acetate membrane electrophoresis into more-anionic forms. Incubation with lectins, proteases, D-glucosidases, neuraminidase, and some effectors did not modify this conversion, which was promoted by rising temperature and pH values. Increasing concentrations of ammonium ions delayed the transformation of amylolytic fractions, thus indicating non-enzymatic deamidation as the reason for amylase isoenzyme development. A change of molecular weight could be excluded. 

A novel p-glucosidase (FPG) from Fusarium proliferatum ECU2042 was successfully purified to homogeneity with a 506-fold increase in specific activity. Its molecular mass was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and its p7 (isoelectric point) was 4.4 as indicated by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C. The enzyme was stable at pH 4.0-6.5 and temperatures below 60 °C, and the inactivation energy ( ) for FPG was 88.6 kj moT. ... 

The extents of sialylation of eleven plasma hydrolases have been examined. Most plasma hydrolases e.g. a-L-fucosidase and a-D-mannosidase) were eluted from DEAE-cellulose at a lower salt concentration after treatment with neuraminidase, although the elution profiles of p-D-glucosidase, P-D-xylosidase, and acid phosphatases were unaffected, indicating that they are less susceptible to the action of neuraminidase. The structure (9) assigned (G. Spick, B. Bayard, B. Fournet, and G. Strecker, F.E.B.S. Letters, 1975, 50, 296) to the carbohydrate component of human serotransferrin has been confirmed by high-resolution H n.m.r. spectroscopy. Electrophoresis separated human serotransferrin into four molecular forms that contain different proportions of bound iron. The nature of the interaction of renins from human and rabbit kidneys with immobilized concanavalin A, and their subsequent desorption with methyl a-D-mannopyrano-side, suggested that both proteins are glycosylated. ... 

Most of the neutral a-D-glucosidases in human liver are bound to particles. Gel filtration and electrophoresis showed that the soluble neutral a-D-glucosidases exist in, at least, four forms with similar pH optima (pH 6.0—6.6) but with different molecular weights (1.1—3.1 x 10 ) and substrate specificities. 

Two isoenzymes (A and B) of j8-D-2-acetamido-2-deoxyglucosidase (mol. wts. 2.0 X 10 and 1.9 x 10 by gel filtration, p/ values 5.31 and 6.78) have been purified to homogeneity on isoelectric focusing from bull seminal plasma. Two identical subunits (mol. wts. 5.3 x 10 and 1.34 x 10 ) were obtained from the A and B isoenzymes when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate or other dissociating agents. The j8-D-2-acetamido-2-deoxy-glucosidase and -galactosidase activities of the two... 

Cellobiose or cellulose were necessary to induce the production of fS-D-glucosidase activity by Sporotrichum pulverulentum. Purification via preparative slab gel isoelectric focusing and affinity chromatography on a phenyl derivative of agarose gave five active fractions (p/ s 4.5—5.2, mol. wts. 1.65—1.82 X 10 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). 

The separation by polyacrylamide gel electrophoresis and subsequent analysis of the components of guinea pig intestinal brush border membrane revealed the presence of inter alia enzyme complexes , a-D-glucosidase-glucoamylase, a-D-glucosidase-sucrose, and a-D-glucohydrolase-glucoamylase. Glucoamylase activity has been solubilized from rat intestinal mucosa (see p. 406). ... 

For experimental details of peptide isolation and sequence analysis, the reader is referred to Volume XXV of this series. However, standard procedures for disulfide reduction, thiol blocking, and tryptic hydrolysis have to be slightly modified, owing to the extreme sensitivity of the bound label in the completely denatured protein or isolated peptides at pH values above 7. This also puts a limit on the conditions of ion-exchange chromatography and electrophoresis. With peptides from /3-glu-cosidase As from A. wentii, the following half-lives (at 26°) were observed. pH 7.0, 30 hr pH 8.6, 5.4 hr pH 9.0, 0.6 hr. However, the label is very stable under acidic conditions, e.g., pH 2.0, aqueous acetic acid up to 25%, 70% formic acid. A similar observation was made with p-glucosidases from sweet almonds and sucrase-isomaltase. ... 

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