Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen

Extraction with Tris–citrate or Tris–NaCl–EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40–50 fold over the previously recommended 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 PLA2 activity was optimum at pH 8.0, heat sensitive at 70°C for 30 min, activated by Ca2+ (although ∼60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage from sperm. PLA2 in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.