title: Purification and characterization of two isoforms of serine proteinase from the microsomal membranes of rat liver.
abstract: An extensive purification of microsomal serine proteinase was achieved from rat liver microsome membranes solubilized with 1% sodium cholate by a series of column chromatographic steps on hydroxylapatite, DEAE-cellulose, and benzamidine-Sepharose 6B, and polyacrylamide gel electrophoresis (PAGE). In the final step of PAGE, the enzyme was separated into two fractions with slightly different mobilities, designated microsomal serine proteinases 1 (MSP1) and 2 (MSP2). The former was purified about 8,000-fold to apparent homogeneity, and the latter was purified about 800-fold, starting from the sample which had been spontaneously activated after elution from the hydroxylapatite column. Both enzyme fractions showed essentially the same properties, including molecular weight, susceptibility to various proteinase inhibitors and metal ions, specificity of action toward protein substrates with special preference for a basic protein histone as well as toward synthetic substrates, and pH dependence of activity toward a synthetic substrate and two neuropeptides. Taken together with the same behavior on a series of chromatographic steps, the two isoforms are thought to be essentially the same enzyme. Further, the detailed kinetic studies of the enzyme activity, especially of MSP1, toward various synthetic and naturally occurring peptide substrates showed that it was highly specific for basic amino acid pairs, strictly hydrolyzing at the COOH side of arginine residue. These results are consistent with those obtained previously with a partially purified enzyme and establish more definitely and in detail the specificity as well as other molecular and enzymatic properties of the enzyme.
authors: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo.
authors: Y
authors: Y
authors: Tamanoue
authors: T
authors: T
authors: Takahashi
authors: K
authors: K
authors: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003374:Coumarins; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D008670:Metals / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D008670:Metals; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C041841:arginine 4-methyl-7-coumarylamide; D001120:Arginine; D012697:Serine Endopeptidases