Thakkar J K, East J, Franson R C
Biol Reprod. 1984 Apr;30(3):679-86. doi: 10.1095/biolreprod30.3.679.
Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.
采用[1-14C]油酸标记的高压灭菌大肠杆菌和1-[1-14C]硬脂酰-2-酰基-3- sn -甘油磷酸乙醇胺作为底物，测定射出的、洗涤后的人类精子超声裂解物和酸提取物中的磷脂酶A2活性。磷脂酶A在pH 7.5时活性最佳，依赖于钙，且仅催化从磷脂2位释放脂肪酸。该活性与膜相关，通过用0.18 N H2SO4萃取可使其溶解。人类精子的酸提取物具有最高的比活性（每毫克1709 nmol/h），其次是小鼠、兔子和公牛，分别为每毫克105、36和1.7 nmol/h。对溴苯甲酰溴抑制人类精子磷脂酶A2活性，但米帕林无作用。在添加1.0 mM CaCl2的情况下，磷脂酶A2活性受到Zn2+和Mn2+抑制；而Cu2+、Cd2+、Mg2+或Sr2+无作用。低浓度（10(-6)至10(-8) M）的Zn2+刺激活性，而在10(-5) M浓度时以剂量依赖方式抑制活性。低浓度Zn2+的刺激程度取决于Ca2+浓度；在10(-7) M时, 0.5 mM CaCl2存在下Zn2+活性被刺激160%，而1.0 mM CaCl2存在下仅被刺激120%。低浓度（10(-5)至10(-7) M）时，甲氧基维拉帕米（D600）和三氟拉嗪刺激人类精子磷脂酶A2活性，三氟拉嗪在10(-5)至10(-4) M药物浓度之间产生几乎完全抑制，而D600无此作用。本文讨论了人类精子磷脂酶A2活性及其受Ca2+、Zn2+和Mn2+调节在精子顶体反应中的意义。
