Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi.
A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.
Saitoh
Y
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Yokosawa
H
H
Takahashi
K
K
Ishii
S
S
D000818:Animals; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D008854:Microscopy, Electron; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis / Q000302:isolation & purification*; D010063:Ovum / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000032:analysis; D013379:Substrate Specificity; D014561:Urochordata / Q000378:metabolism*
D006882:Hydroxyapatites; D009097:Multienzyme Complexes; D011506:Proteins; D017886:Durapatite; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex