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Characterization and inhibitor sensitivity of human sperm phospholipase A 2 : Evidence against pivotal involvement of phospholipase A 2 in the acrosome reaction
Author(s) -
Anderson Jr. Robert A.,
Johnson Susan K.,
Bielfeld Peter,
Feathergill Kenneth A.,
Zaneveld Lourens J. D.
Publication year - 1990
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080270405
Subject(s) - acrosome reaction , lysophosphatidylcholine , phospholipase a2 , biology , phospholipase a , calmodulin , enzyme , enzyme assay , non competitive inhibition , dissociation constant , trifluoperazine , biochemistry , sodium , phosphatidylcholine , stereochemistry , chemistry , in vitro , phospholipid , receptor , organic chemistry , membrane
The kinetic properties and inhibitor sensitivity of human sperm phospholipase A 2 (PLA 2 ; 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 Ca 2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA 2 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 PLA 2 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 Ca 2+ dependent and heat stable; however, the low‐K m activity was less resistant to 60°C preincubation at pH 7.5 (28% inactivation of low‐K m activity after 45 min, as compared to no effect on high‐K m activity). Quinacrine was a noncompetitive PLA 2 inhibitor with K i s for low‐ and high‐K m activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high‐K m activity noncompetitively (K i = 87 μM) and the low‐K m activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low‐ and high‐K m activities by p‐bromophenacyl bromide were 0.28 mM and 0.032 min −1 , and 0.73 mM and 0.066 min −1 , respectively. PLA 2 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%) PLA 2 at concentrations from 2–5 mM but inhibited PLA 2 (40–50%) at a concentration of 10 mM. MnCl 2 (5mM) inhibited low‐ and high‐K m PLA 2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 μM), p‐bromophenacyl bromide (20 μM), and MnCl 2 (5 mM) were tested as inhibitors of the ionophore A23187‐induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl 2 (93%). The effect of MnCl 2 was restricted to an interaction with A23187, rather than with PLA 2 ; p‐Bromophenacyl bromide inhibited (P < 0.05) PLA 2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA 2 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 PLA 2 , with properties similar to those of PLA 2 in other species and tissues; (2) PLA 2 inhibition is not a likely mechanism for inhibition of acrosome reactions by acrosin inhibitors; (3) sperm PLA 2 may be modulated by an endogenous activator; (4) the low‐K m PLA 2 is similar to the group I (pancreatic) enzyme; and (5) PLA 2 is not a pivotal or rate‐limiting step in the human acrosome reaction.