Human serum dopamine-beta-hydroxylase: purification, molecular weight, presence of sugars and kinetic properties.
1) Dopamine-beta-hydroxylase has been purified from human serum. Ammonium sulphate fractionation was used as the first step, the enzyme being precipated between 30 and 50 percent saturation. For the second step, the enzyme was adsorbed onto, and then eluted from a column of Concanavalin A covalently bound to Sepharose 4B. In the third step the enzyme was further purified by passage through two successive columns of Sephadex G-200. The final step, chromatography on DEAE-cellulose, gave a preparation of the enzyme with a specific activity of 36 mumoles of octopamine formed/30 min per mg of enzyme, representing a purification from the starting serum of 3,000 fold. 200 mug of enzyme could be obtained from 200 ml of serum. 2) The enzyme preparation was found to be pure or, at most, only slightly contaminated (depending on the starting serum) as judged by the criterion of polyacrylamide gel electrophoresis. 3) No cross-reaction could be observed between the human serum enzyme and rabbit antibody against bovine adrenal dopamine-beta-hydroxylase. 4) The molecular weight of the enzyme was found to be 250,000, i.e. less than the bovine adrenal enzyme. 5) Kinetic properties of the enzyme were studied. The mechanism of action of the enzyme was found to be similar to that of the bovine adrenal enzyme. Real kinetic parameters for both tyramine and ascorbate were calculated and found to be the same as those described for the bovine adrenal enzyme. 6) Interaction with Concanavalin A strongly suggests that the serum dopamine-beta-hydroxylase is a glycoprotein.
D001205:Ascorbic Acid / Q000494:pharmacology; D003208:Concanavalin A; D004299:Dopamine beta-Hydroxylase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D014439:Tyramine / Q000494:pharmacology
D006023:Glycoproteins; D003208:Concanavalin A; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid; D014439:Tyramine