Multicatalytic proteinase in fish muscle.
Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.
Folco
E J
EJ
Instituto Nacional de Tecnología Industrial, Centro de Investigaciones de Tecnología Pesquera, Mar del plata, Argentina.
Busconi
L
L
Martone
C B
CB
Sanchez
J J
JJ
D000818:Animals; D002845:Chromatography / Q000379:methods; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005227:Fatty Acids / Q000494:pharmacology; D005399:Fishes; D006868:Hydrolysis; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009132:Muscles / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000032:analysis; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D003902:Detergents; D005227:Fatty Acids; D009097:Multienzyme Complexes; D011506:Proteins; D013438:Sulfhydryl Compounds; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex