Single active site mechanism of rabbit liver acid alpha-glucosidase.
An acid alpha-glucosidase was purified from rabbit liver by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-Toyopearl, Toyopearl HW-55F, and Toyopearl HW-65F column. The resulting preparation showed a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be 1.03 X 10(4) by SDS-disc electrophoresis. The optimum pH was found to be 4.7. The alpha-glucosidase showed relatively high activity not only toward maltose but also toward alpha-glucans, such as shellfish glycogen, soluble starch, beta-limit dextrin, amylopectin, and amylose. The Km values for maltose and shellfish glycogen were 2.1 and 16 mM (the concentration of non-reducing glucose units), respectively, and the ratio of maximum velocities of hydrolysis of the two substrates was 100:133. The nature of the active site catalyzing the hydrolyses of maltose and shellfish glycogen was investigated by electrophoresis in the presence of urea and by kinetic methods. The purified enzyme was not separated into two components, maltase and glycogen hydrolase, in the electrophoretic gel containing 3 M urea, contrary to the report by Belenki and Rosenfeld ((1972) Biochem. Biophys. Res. Commun. 46, 443-448). In experiments with mixed substrates of maltose and glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, Mg2+, were about equally effective for the stimulation of enzyme action on maltose and glycogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Onodera
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Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University.
Matsui
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Chiba
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D000645:Ammonium Sulfate; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006003:Glycogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008320:Maltose / Q000378:metabolism; D008970:Molecular Weight; D011817:Rabbits; D014508:Urea / Q000494:pharmacology; D000520:alpha-Glucosidases / Q000302:isolation & purification / Q000378:metabolism*
D008320:Maltose; D014508:Urea; D006003:Glycogen; D000520:alpha-Glucosidases; D000645:Ammonium Sulfate