Folco E J, Busconi L, Martone C B, Sanchez J J
Instituto Nacional de Tecnología Industrial, Centro de Investigaciones de Tecnología Pesquera, Mar del plata, Argentina.
Arch Biochem Biophys. 1988 Dec;267(2):599-605. doi: 10.1016/0003-9861(88)90067-7.
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.
蛋白酶II是一种先前在白姑鱼骨骼肌中鉴定出的高分子量蛋白酶，通过DEAE-琼脂糖凝胶、苯基琼脂糖凝胶CL 4B和Sephacryl S-300色谱法纯化至表观均一。在变性条件下，该酶解离成一组亚基，其Mr范围为18,000至26,000，以及一个Mr为60,000的大亚基。该蛋白酶能够水解在荧光基团附近具有芳香族氨基酸（类胰凝乳蛋白酶活性）或精氨酸残基（类胰蛋白酶活性）的N端封闭的4-甲基-7-香豆素酰胺底物。该酶的类胰蛋白酶活性受到脂肪酸和十二烷基硫酸钠的抑制，而类胰凝乳蛋白酶活性则受到这些化合物的刺激，但受到非离子和阳离子去污剂的抑制。几种巯基试剂抑制了蛋白酶II的两种活性。然而，亮肽素和Cu2+强烈抑制其类胰蛋白酶活性，但仅轻微影响其类胰凝乳蛋白酶活性。二硫苏糖醇刺激了两种活性，但程度不同且在不同的浓度范围内。这些结果表明该酶具有多催化性，至少有两个不同的活性位点。
