title: Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium.
abstract: An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.
authors.affiliation: Middle East Technical University, Department of Biological Sciences, Ankara, Turkey. biosemra@metu.edu.tr
authors.forename: Semra
authors.initials: S
authors.lastname: Kocabiyik
authors.forename: Inci
authors.initials: I
authors.lastname: Ozdemir
mesh_terms: D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D002413:Cations, Divalent; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D008274:Magnesium; D002118:Calcium