Characterization and inhibitor sensitivity of human sperm phospholipase A2: evidence against pivotal involvement of phospholipase A2 in the acrosome reaction.
The 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 microM and 0.64 mlU/mg protein and the other with respective constants of 630 microM 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 degrees 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 microM) 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 microM), p-bromophenacyl bromide (20 microM), 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Anderson
R A
RA
Department of Obstetrics and Gynecology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois.
Johnson
S K
SK
Bielfeld
P
P
Feathergill
K A
KA
Zaneveld
L J
LJ
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000098:Acetophenones / Q000494:pharmacology; D000177:Acrosome / Q000502:physiology*; D000328:Adult; D002118:Calcium / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011796:Quinacrine / Q000494:pharmacology; D013094:Spermatozoa / Q000201:enzymology*; D013696:Temperature; D014268:Trifluoperazine / Q000494:pharmacology
D000098:Acetophenones; D008244:Lysophosphatidylcholines; D003840:Deoxycholic Acid; D014268:Trifluoperazine; D015060:1,2-Dipalmitoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2; D011796:Quinacrine; C006821:4-bromophenacyl bromide; D002118:Calcium; D004121:Dimethyl Sulfoxide