Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi.
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 strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.
Shishikura
F
F
Department of Biology, Nihon University School of Medicine, Tokyo, Japan. fshishi@med.nihon-u.ac.jp
Abe
T
T
Ohtake
S
S
Tanaka
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D006458:Hemolymph / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014561:Urochordata / Q000201:enzymology*
D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D005340:Fibrinogen; D012697:Serine Endopeptidases