Purification and characterization of a novel acrosin-like enzyme from boar cauda epididymal sperm.
A trypsin-like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted 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 protein 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 pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50 mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kcat/Km) 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 sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.
Akama
K
K
Department of Chemistry, Faculty of Science, Chiba University.
Terao
K
K
Tanaka
Y
Y
Noguchi
A
A
Yonezawa
N
N
Nakano
M
M
Tobita
T
T
D000176:Acrosin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002845:Chromatography / Q000379:methods; D004822:Epididymis / Q000166:cytology; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine
D002593:Cetrimonium Compounds; D012697:Serine Endopeptidases; D000176:Acrosin; D000077286:Cetrimonium