Identification and localization of a novel cathepsin S-like proteinase in guinea pig spermatozoa.
Guinea pig spermatozoa were found to contain a novel cysteine proteinase that most closely resembled lysosomal cathepsin S. The responsible enzyme was shown to have an acrosomal localization. Like the cathepsin S of lymphocyte-rich tissues, e.g., lymph nodes and spleen, the spermatozoal enzyme digested protein substrates such as hemoglobin and Azocoll optimally at about pH 3.5 in the presence of pepstatin. No appreciable action was manifested in the range of pH 5-6 on protein substrates nor on the 7-(4-methyl)coumarylamide (NMec) derivatives of peptides commonly employed for the fluorometric assay of cathepsins B (benzyloxycarbonyl (Z)-Arg-Arg-NMec), H (Arg-NMec), and L (Z-Phe-Arg-NMec). Like spleen cathepsin S, the sperm enzyme selectively hydrolyzed Z-Phe-Arg-NMec, but its activity was uncharacteristically limited to a pH range of 3.0-3.5, where it displayed a typical high sensitivity to sulfhydryl reagents (HgCl2, mersalyl acid, iodoacetate), leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxysuccinylleucylamido(3-methyl)butane. Gossypol (a male antifertility agent) was also inhibitory. Inhibition by the mercurial sulfhydryl reagents was completely reversible with dithiothreitol. Pepstatin, a potent inhibitor of aspartic proteinases, and serine proteinase inhibitors (phenylmethylsulfonyl fluoride, benzamidine) were ineffective. Other properties displayed by the sperm extract that were uniquely characteristic of cathepsin S included stability under alkaline conditions, and a greater rate of hydrolysis when the P2-phenylalanine of the assay substrate was replaced by two aliphatic residues, as in Z-Leu-Leu-Arg-NMec.
McDonald
J K
JK
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Culbertson
J T
JT
Owers
N O
NO
D000177:Acrosome / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001391:Azo Compounds / Q000378:metabolism; D002244:Carbon; D002403:Cathepsins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D004396:Coloring Agents; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D005780:Gelatin / Q000378:metabolism; D006168:Guinea Pigs; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001391:Azo Compounds; D004396:Coloring Agents; D005456:Fluorescent Dyes; D006454:Hemoglobins; D010436:Pepstatins; D010455:Peptides; D013439:Sulfhydryl Reagents; C028433:chinese ink; C014195:Streptomyces pepsin inhibitor; C028470:Azocoll; D002244:Carbon; D005780:Gelatin; D003094:Collagen; D002403:Cathepsins; C028217:cathepsin S; D004229:Dithiothreitol; C031375:pepstatin