Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.
We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin.
Adeniran
A J
AJ
Department of Physiology and Biophysics, School of Medicine, Health Sciences Center State University of New York, Stony Brook 11794-8661, USA.
Shoshani
I
I
Minuth
M
M
Awad
J A
JA
Elce
J S
JS
Johnson
R A
RA
D000176:Acrosin / Q000737:chemistry* / Q000302:isolation & purification; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D013094:Spermatozoa / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D000911:Antibodies, Monoclonal; D010761:Phosphorus Radioisotopes; C045523:ninhibin; D000176:Acrosin; D000262:Adenylyl Cyclases