Studies on the measurement of phospholipase A(2) (PLA(2)) and PLA(2) inhibitor activities in ram semen

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phosph olipase A(2) (PLA(2)) from ram semen by 40-50 fold over the previously reco mmended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl -EGTA. The semen PLA(2) activity was optimum at pH 8.0, heat sensitive at 7 0 degrees C for 30 min, activated by Ca2+ (although similar to 60% activity was also found in the absence of calcium) and did not exist as a pro-enzym e. The semen PLA(2) activity was equally distributed among the sperm and se minal plasma (SP) components of ram semen. However, the low levels of PLA(2 ) activity in the SP of vasectomised rams tend to suggest that PLA(2) in th e SP fraction may have originated from testicular or epididymal secretions or leakage from sperm. PLA(2) in sperm exists as a large molecular weight a ggregate, whereas in SP it is present as a smaller aggregate. In addition t o PLA(2), semen also contained PLA(2) inhibitor activities. Inhibition was observed against PLA(2)s from bee venom, pig pancreas and oviductal extract s. The inhibitory activity is presumed to be due to a large molecular weigh t protein as the inhibitor activity was not extracted in a chloroform:metha nol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichlor oacetic acid and destroyed by proteases. The inhibitor activity was distrib uted in various molecular weight fractions of sperm, SP and SP from vasecto mised rams. (C) 1999 Elsevier Science B.V. All rights reserved.