Manjunath P, Soubeyrand S, Chandonnet L, Roberts K D
Department of Medicine, University of Montreal, Quebec, Canada.
Biochem J. 1994 Oct 1;303 ( Pt 1)(Pt 1):121-8. doi: 10.1042/bj3030121.
We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.
我们最近发现，牛精浆中的主要蛋白质，即BSP-A1、BSP-A2、BSP-A3和BSP-30-kDa（统称为BSP蛋白）可与精子结合，精子质膜上的结合位点是胆碱磷脂。鉴于这些磷脂是磷脂酶A2（PLA2）的底物，而磷脂酶A2是精子获能和顶体反应中的关键酶，因此研究了BSP蛋白对该酶活性的影响。由于这些BSP蛋白普遍存在，所以也研究了其对猪胰PLA2的影响。与对照蛋白相反，当以磷脂酰胆碱为底物进行预孵育时，所有BSP蛋白均以剂量依赖的方式抑制胰PLA2和精子PLA2的活性，在存在1 - 6 microM BSP蛋白时，酶活性完全被消除。当以磷脂酰乙醇胺为底物时，仅胰PLA2受到抑制。另一方面，当BSP蛋白与酶预孵育后再添加底物时，观察到双相效应；在BSP浓度低于1.3 microM时酶活性受到刺激，高于此浓度则受到抑制。抑制活性对胰蛋白酶敏感但耐热。研究了参与精子获能且也与BSP蛋白相互作用的肝素共同孵育的影响。肝素（10 microM）对所有BSP蛋白表现出的PLA2抑制活性没有影响。BSP蛋白表现出的PLA2抑制作用可被过量底物消除。BSP蛋白吸附在PLA2 - 琼脂糖上，并可与该酶进行亲和交联，表明酶与抑制剂之间存在直接相互作用。这些结果表明，这些BSP蛋白调节PLA2活性，进而调节磷脂代谢。
