Purification of a novel phospholipase A2 from bovine seminal plasma.
Phospholipases A2 are enzymes believed to play important roles in numerous physiological systems including sperm cell maturation. Relatively little work has, however, been devoted to study these enzymes in seminal plasma. We therefore undertook the purification and characterization of this enzyme from bovine seminal plasma. After a 330-fold purification, an activity corresponding to a protein of 100 kDa was identified by gel filtration. SDS-polyacrylamide gel electrophoresis analysis of the purified fraction revealed the presence of a 60-kDa band that comigrated with the activity during ion-exchange and gel filtration chromatography as well as polyacrylamide gel electrophoresis. The enzyme possessed a pH optimum around pH 6.5 and was calcium-dependent. Using isoelectric focusing, its isoelectric point was determined to be 5.6 +/- 0.07. The enzymatic activity was resistant to p-bromophenacyl bromide, but was sensitive to gossypol and dithiothreitol. The enzyme was 2 orders of magnitude more active toward micelles formed with deoxycholate than with Triton X-100. Slight differences in the specificity toward head groups and/or sn-2-side chains were found in both assay systems. The enzyme was acid-labile and did not display affinity for heparin. It would therefore appear that the phospholipase A2 form isolated from bovine seminal plasma is of a novel type.
Soubeyrand
S
S
Department of Medicine, University of Montreal, Quebec, Canada.
Khadir
A
A
Brindle
Y
Y
Manjunath
P
P
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006072:Gossypol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D054467:Phospholipases A2; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine
D000098:Acetophenones; D004791:Enzyme Inhibitors; D010741:Phospholipases A; D054467:Phospholipases A2; D006072:Gossypol; C006821:4-bromophenacyl bromide; D002118:Calcium