Purification of benzylamine oxidase from cultured porcine aortic smooth muscle cells.
Soluble benzylamine oxidase (BzAO) from cell homogenates and the conditioned culture medium of porcine aortic smooth muscle cells was purified by anionic HPLC methods and characterized with regard to enzyme kinetics and inhibition by semicarbazide, phenelzine, cuprizone, diethyldithiocarbamate (DDC), and p-chloromercuriphenylsuphonate (PCMPS). BzAO from both the cell homogenates and the conditioned culture medium had an Mr of 130,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using [methylene-14C]benzylamine hydrochloride as substrate, BzAO from cell homogenates and the conditioned culture medium had Km values of 5.1 and 6.1 microM, respectfully, and Vmax values of 89 and 53 nmol.mg protein-1.h-1. Both enzymes were sensitive to inhibition by semicarbazide and phenelzine, but insensitive to inhibition by the copper chelating agent DDC. BzAO isolated from the conditioned culture medium was more sensitive to inhibition by lower concentrations of cuprizone and PCMPS than the enzyme isolated from cell homogenates. Antisera raised against BzAO from cell homogenates reacted with BzAO from the conditioned culture medium and from porcine plasma.
Hysmith
R M
RM
Department of Pathology, University of Texas Medical Branch, Galveston 77550.
Boor
P J
PJ
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000201:enzymology; D001595:Benzylamine Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D003471:Cuprizone / Q000494:pharmacology; D004050:Ditiocarb / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008970:Molecular Weight; D008995:Monoamine Oxidase / Q000302:isolation & purification*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D012642:Selegiline / Q000494:pharmacology; D012663:Semicarbazides / Q000494:pharmacology; D013552:Swine
D002793:Cholic Acids; D012663:Semicarbazides; D012642:Selegiline; C010059:carbamylhydrazine; D003471:Cuprizone; D002731:4-Chloromercuribenzenesulfonate; D004050:Ditiocarb; D001595:Benzylamine Oxidase; D008995:Monoamine Oxidase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate