Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
F ShishikuraK Tanaka

Abstract

A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and str...Continue Reading

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Citations

Nov 13, 2008·Cytotechnology·Ronaldo Z MendonçaCarlos A Pereira
Jun 7, 2000·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·N GuerrieriP Cerletti
Jul 19, 2001·Proceedings of the National Academy of Sciences of the United States of America·J SheehanP Jagadeeswaran
Oct 18, 2006·Proceedings of the National Academy of Sciences of the United States of America·John D KulmanEarl W Davie
Jan 13, 2006·Journal of Invertebrate Pathology·Loriano Ballarin, Paolo Burighel
Jul 18, 2018·The Journal of Experimental Biology·Neha Acharya-PatelWilliam K Milsom
Apr 25, 2019·The Journal of Experimental Biology·Matthew J H GilbertAnthony P Farrell
Oct 2, 2007·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Yu XinRongxiu Li
Oct 30, 2007·Developmental Biology·Yuval RinkevichRam Reshef

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