Purification and characterization of a 58,000-Da proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi.
A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.
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; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D011232:Chemical Precipitation; D002850:Chromatography, Gel / Q000379:methods; D016264:Dextran Sulfate / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004798:Enzymes / Q000097:blood / Q000187:drug effects; D006458:Hemolymph / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014561:Urochordata / Q000737:chemistry*
D000596:Amino Acids; D004798:Enzymes; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D016264:Dextran Sulfate