JBC, Vol. 253, Issue 23, 8428-8432, Dec, 1978
Boar malpha-acrosin. Purification and characterization of the inital active enzyme resulting from the conversion of boar proacrosin to acrosin
R. F. Parrish and K. L. Polakoski
The preparation of highly purified malpha-acrosin is described.
Purification was achieved by controlled activation of partially purified
proacrosin, followed by gel chromatography over Sephadex G-100 at pH 3.0.
The final malpha-acrosin preparation resulted in a single protein band with
a molecular weight of 49,000 as determined by sodium dodecyl sulfate-disc
gel electrophoresis. Disc arginine naphthylamide hydrolyzing band with a
relative migration of 0.39 malpha-acrosin catalyzed the hydrolysis of
synthetic substrates containing arginine and lysine, but not phenylalanine.
Although calcium ions were not required for enzymatic activity, the
addition of calcium chloride stimulated the activity through an increased
substrate affinity and an increased maximal velocity. Polyamines stimulated
the maximal velocity of the reaction, but were without effect on the
substrate affinity. malpha-Acrosin was inhibited by lima bean, ovo-mucoid,
and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and
1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an
irreversible inhibition, while L-arginine, benzamidine, and
p-aminobenzamidine were competitive inhibitors with respect to substrate.
These properties of malpha-acrosin are very similar to those previously
reported for mbeta-acrosin and suggest that the portion of the molecule
lost during the conversion of malpha-acrosin to mbeta-acrosin contributes
little to the topography of either the active site or regulatory sites of
the enzyme.
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