K. Akama et al., PURIFICATION AND CHARACTERIZATION OF A NOVEL ACROSIN-LIKE ENZYME FROMBOAR CAUDA EPIDIDYMAL SPERM, Journal of Biochemistry, 116(2), 1994, pp. 464-470
A trypsin-like protease was extracted with 1% cetyltrimethylammonium b
romide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose
acrosin had previously been removed by acid extraction. The CTAB-extra
cted sperm protease (CSP) was purified by ion-exchange chromatography
on CM-23, gel filtration on Sephadex G-100, affinity chromatography on
benzamidine-CH-Sepharose 4B, and HPLC on CM-5PW. CSP is a two chain p
rotein composed of M(r) 2.6K and M(r) 37K chains, which are covalently
cross-linked by disulfide bonds. CSP exhibited a pH optimum between p
H 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, a
ntipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. T
he activity of CSP was enhanced about 1.2-fold with 50 mM CaCl2 with w
hich acrosin is enhanced 2.0-fold. The catalytic efficiency (k(cat)/K-
m) of CSP toward Bz-L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the
presence of 50 mM CaCl2 differed from that of acrosin by factors of 0
.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested
peptides of CSP, and its L- and H-chains showed that the amino acid s
equence of CSP was closely related to, but different from, that of acr
osin. These results suggest that CSP is a novel acrosin-like enzyme th
at differs from acrosin in its location in the sperm head, the effect
of calcium ions on its activity, and its substrate specificity.