| Abstrakt: |
Dopamine β‐hydroxylase (DβH) (EC 1.14.17.1) from adrenal medulla is a glycoprotein with approximately 5% carbohydrate by weight. The oligosaccharide chains of this enzyme were enzymatically removed with various glycosidic enzymes (endoglycosidases D, F, and H; glycopeptidase F; α‐mannosidase; neuraminidase; and β‐galactosidase). The time course of deglycosylation was monitored by polyacrylamide gel electrophoresis, and evidence for sugar removal was shown by a modification of the Western blot technique utilizing 125I‐labeled concanavalin A and by amino acid analysis. Protein was detected in the gel by using specific antibodies and 125I‐labeled protein A. Steady‐state kinetic data of deglycosylated DβH show minor differences between the native and the deglycosylated protein. The Kmvalues for tyramine were 2.17 and 1.66 mmwhereas the Kmvalues for oxygen were 0.18 and 0.14 mmfor the native and the deglycosylated protein, respectively. The Vmaxvalues (pH 5.0) for the two forms of the enzyme were comparable, with the deglycosylated DβH being 15% lower. These data indicate that the oligosaccharide moieties present on DβH do not play a role in catalysis.— Hamos, J.; Desai, P. R.; Villafranca, J. J. Characterization and kinetic studies of deglycosylated dopamine β‐hydroxylase. FASEB J.1: 143‐148; 1987. |
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