Citation

Yabanoǧlu S, Uçar G. Fabad Journal of Pharmaceutical Sciences 2005; 30(2): 64-77.

Copyright

(Copyright © 2005)

DOI

unavailable

PMID

unavailable

Abstract

Semicarbazide-sensitive amine oxidase (SSAO), an enzyme involved in detoxifying xenobiotics, regulating glucose uptake, and effecting cell adhesion, leukocyte trafficking and angiogenesis, was purified from the crude microsomal fractions of rat lung by Cibacron Blue 3GA-agarose and Concanavalin A-Sepharose 4B affinity chromatographies with a specific activity of 5.554 nmol/min/mg of protein. A 46-fold purification and a recovery of about 15% were obtained. Purity was approved by polyacrylamide gel electrophoresis (PAGE). The purified SSAO appeared as a single band with a molecular mass of 184 kDa in PAGE; however, sodium dodecyl sulfate (SDS)-PAGE under reducing conditions yielded a band of 93 kDa, suggesting that the enzyme is a homodimer which is composed of 93 kDa subunits possibly attached by disulfide bridges. Optimum temperature and pH were found as 45°C and 7.5, respectively. Incubation at 60°C resulted in a complete loss of SSAO activity. Purified SSAO was stable for at least one month at -80°C. Km and Vmax values towards its substrate benzylamine were determined to be 3.65 μM and 5.6 nmol/min/mg, respectively. The velocity of the reaction decreased with increasing substrate concentration when benzylamine was used as substrate, indicating that Michaelis-Menten enzyme behavior was obeyed at only low concentrations of the substrate. Semicarbazide exhibited a suicide type of inhibition on the oxidation of benzylamine by SSAO by possibly interacting first with the enzyme to form a reversible enzyme-inhibitor complex with a subsequent reaction and then leading this complex to the covalently bound enzyme-inhibitor adduct.

Language: en

Keywords

male; article; controlled study; Purification; nonhuman; enzyme activity; oxidation; temperature measurement; enzyme inhibition; enzyme kinetics; enzyme substrate; rat; enzyme analysis; enzyme stability; amine oxidase (copper containing); animal tissue; benzylamine; disulfide; enzyme purification; enzyme specificity; enzyme subunit; homodimer; incubation time; Inhibition; lung parenchyma; Michaelis Menten kinetics; microsome; molecular weight; pH measurement; polyacrylamide gel electrophoresis; Rat lung; semicarbazide; Semicarbazide-sensitive amine oxidase (SSAO); Substrate; velocity