GUINEA-PIG TESTICULAR PROACROSIN-ACROSIN SYSTEM - FURTHER CHARACTERIZATION OF THE ACTIVE ENZYME
GUINEA-PIG TESTICULAR PROACROSIN-ACROSIN SYSTEM - FURTHER CHARACTERIZATION OF THE ACTIVE ENZYME
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DOI：
10.1095/biolreprod40.1.127
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发表时间：
1989-01-01
影响因子：
3.6
通讯作者：
TEUSCHER, C
中科院分区：
文献类型：
--
作者：
ADEKUNLE, AO;STOREY, BT;TEUSCHER, C
In this paper, the characteristics of a highly stable, 34,000 molecular weight form of guinea pig (GP) acrosin are compared with those of acrosins from other mammalian species. GP acrosin, like acrosins from other species, is stable at pH 3.0, a pH optimum of 8.0, and is inhibited by natural trypsin inhibitors and N-.alpha.-p-tosyl-L-lysine chloromethyl ketone. Its lack of inhibition by tosyl-phenylalanine chloromethyl ketone indicates that is has a specificity similar to trypsin but not chymotrypsin. The activity of GP acrosin was stimulated by Ca2+ below 75 mM. The enzyme was markedly inhibited by Hg2+, but only weakly inhibited by other metal cations. The disulfide reductants dithiothreitol and 2-mercaptoethanol both inhibited GP acrosin, as did the sulfhydryl reactant, iodoacetic acid. the Michaelis-Menten constant for GP testicular acrosin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoromethylcoumarin at pH 8.0 was calculated from Lineweaver-Burk plots to give a value of Km = 2.0 .times. 10-5 M with Vmax = 500 .mu.moles/min/mg protein. The corresponding lysine substrate, the N-benzyloxy-carbonyl L-lysine amide of 7-amino-4-trifluoromethyl-coumarin, had a higher Km = 4.6 .times. 10-5 M and lower Vmax = 135 .mu.moles/min/mg protein, in accord with the substrate preference seen with other mammalian acrosins. GP acrosin differs from other mammalian acrosins in that it exhibits weak activation by Ca2+, is active in the presence of ethylene glycol-bis (.beta.-amino-ethyl ether) N,N,N''N''-tetraacetic acid (EGTA) and does not demonstrate an expected "burst" of product in a single enzyme turnover with the activesite titant 4-methylumbelliferyl-p-guanidinebenzoate. Although the functional significance of these differences exhibited by GP acrosin is not known, it is suggested that the response of enzyme activity to Ca2+ and to guanidinobenzoate ester active site titrants may provide a means of differentiating acrosins of different mammalian species.
