Characterization and inhibitor sensitivity of human sperm phospholipase A2: Evidence against pivotal involvement of phospholipase A2 in the acrosome reaction
Authors: Anderson Jr. Robert A. , Johnson Susan K. , Bielfeld Peter , Feathergill Kenneth A. , Zaneveld Lourens J. D. 
Published: February 2005

AbstractThe kinetic properties and inhibitor sensitivity of human sperm phospholipase A2 (PLA2; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn‐2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 mM lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA2 was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCl). Two molecular forms of PLA2 were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 μM and 0.64 mlU/mg protein and the other with respective constants of 630 μM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low‐Km activity was less resistant to 60°C preincubation at pH 7.5 (28% inactivation of low‐Km activity after 45 min, as compared to no effect on high‐Km activity). Quinacrine was a noncompetitive PLA2 inhibitor with Kis for low‐ and high‐Km activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high‐Km activity noncompetitively (Ki = 87 μM) and the low‐Km activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low‐ and high‐Km activities by p‐bromophenacyl bromide were 0.28 mM and 0.032 min−1, and 0.73 mM and 0.066 min−1, respectively. PLA2 was inhibited by p‐nitrophenyl‐p′‐guanidinobenzoate, at higher concentrations (10−4–10−3 M) than required to inhibit trypsinlike proteinases; p‐aminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA2 at concentrations from 2–5 mM but inhibited PLA2 (40–50%) at a concentration of 10 mM. MnCl2 (5mM) inhibited low‐ and high‐Km PLA2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 μM), p‐bromophenacyl bromide (20 μM), and MnCl2 (5 mM) were tested as inhibitors of the ionophore A23187‐induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl2 (93%). The effect of MnCl2 was restricted to an interaction with A23187, rather than with PLA2; p‐Bromophenacyl bromide inhibited (P < 0.05) PLA2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA2 inhibition was poorly correlated with the acrosome reaction. Results from this study suggest that (1) human sperm contain at least two kinetically distinguishable forms of PLA2, with properties similar to those of PLA2 in other species and tissues; (2) PLA2 inhibition is not a likely mechanism for inhibition of acrosome reactions by acrosin inhibitors; (3) sperm PLA2 may be modulated by an endogenous activator; (4) the low‐Km PLA2 is similar to the group I (pancreatic) enzyme; and (5) PLA2 is not a pivotal or rate‐limiting step in the human acrosome reaction.