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Purification and properties of an NADP+-dependent glycerol dehydrogenase from rabbit skeletal muscle

1972; Elsevier BV; Volume: 258; Issue: 1 Linguagem: Inglês

10.1016/0005-2744(72)90965-5

1878-1454

Alfred W. Kormann, R. O. Hurst, T.G. Flynn,

Diet, Metabolism, and Disease

1.|Glycerol dehydrogenase from rabbit skeletal muscle has been isolated and some properties of the enzyme have been determined to gain information about its identity and its biological significance. 2.|The enzyme has been purified 300-fold by (NH4)2SO4 precipitation, gel filtration on Sephadex G-100 and chromatography on DEAE-Sephadex A-50. 3.|Formation of glycerol and NADP+ is strongly favoured resulting in an apparent thermodynamic equilibrium constant of 1.7 · 10−6 for the enzyme at pH 7.0 and 37°. 4.|Molecular weight for the enzyme as determined by gel filtration was 34 000 ± 1000. 5.|Using the glycerol dehydrogenase prepared in this study a method has been determined for assaying the amount of d-glyceraldehyde in solution. 6.|d-Glyceraldehyde was found to be the preferred substrate but the enzyme also catalyzed the reduction of other aldehydes with close structural similarity. Dihydroxyacetone, aldoses and ketoses with 5 or 6 carbon atoms were poor substrates. The activity of the enzyme with NADH as cofactor was 10–12% of the activity with NADPH as cofactor. 7.|The affinity of the enzyme towards reduced compounds was demonstrated on acrylamide gels by applying a technique of combining zymogram and protein stain. The results confirmed the findings of the assays with the corresponding aldehydes. 8.|Enzyme activity was almost completely inhibited by 1 mM p-chloromercuribenzoate and to 84% by 4 M urea at 25°. The presence of 0.5 M (NH4)2SO4 decreased enzyme activity by one-third. 9.|Comparison with other enzymes led to the conclusion that glycerol dehydrogenase from rabbit skeletal muscle has its own identity and that this enzyme should be classified as “glycerol:NADP+ oxidoreductase (d-glyceraldehyde forming)”.