Characterization and inhibitor sensitivity of human sperm phospholipase A₂: Evidence against pivotal involvement of phospholipase A₂ in the acrosome reaction

Abstract

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)

A Basic 18-Amino Acid Peptide Contains the Polysulfate-Binding Domain Responsible for Activation of the Boar Proacrosin/Acrosin System1

“…The data in the literature suggest that the binding of polysulfates to the PSBD of proacrosin stimulates the rate of proacrosin activation. On the other hand, the binding of polysulfates to the PSBD of ␤-acrosin induces inhibition of the enzyme activity [12,23,64,65]. This evidence prompted us to search in the proacrosin sequence for a heparinbinding consensus sequence of the type BBBXXB or BBXB (where X represents any hydropathic amino acid and B any basic amino acid) [60].…”

Superoxide anion production by human spermatozoa as a part of the ionophore‐induced acrosome reaction process

“…Superoxide anion produced by the cells is able to move across the membrane through the anion channels (Lynch & Fridovich, 1978;R o o s et a[., 1984) and acts as a phospholipase inside the membrane (Deby et al, 1990) where it leads in part to the accumulation of lysophospholipids, notably lysophosphatidylcholine (Kinnaird et al, 1988), a fusogenic phospholipid implicated in the acrosome reaction (Fleming & Yanagimachi, 1981;O h z u & Yanagimachi, 1982). Superoxide anion could also enhance the release of lysolipids through activation of the phospholipase A, (Sawada & Carlson, 1991;Carlson & W u , 1991), an enzyme also implicated in the acrosome reaction (Langlais & Roberts, 1985), although its activity may not be rate limiting (Anderson et al, 1990). In fact, de-esterification and membrane fluidity seem related since electron paramagnetic resonance spectrometry revealed that membrane fluidity of erythrocytes was increased after exposure t o tetramethylammonium superoxide, a phenomenon inhibited by the presence of SOD (Rosen et al, 1983).…”

Phospholipase A2 activity in fresh and cryopreserved spermatozoa from boars

“…Of the temperatures examined, 35oC provided maximal activity, paralleling the common selection of 35-40"C for other assays of PLA2 (Thakkar et al 1983(Thakkar et al , 1984Langlais and Roberts 1985: Hinkovska et al 1987: Guerette et al 1988). Those same studies and Anderson et al (1990) …”

“…Artificial insemination with cryopreserved boar semen results in relatively low conception rates and reduced numbers ofpigs per litter (Johnson et al 1981;Johnson 1985) and the effects ofrapid semen cooling and exposure of boar spermatozoa to PLA2 were similar (Robertson et al 1990 Thakkar et al (1984). The washed spermatozoa pellets were resuspended in 0.18 N HISO4 to a concentration of 3 x 10v-4.5 x 10v cells ml-t (total volume 8-17 (Anderson et al 1990). Also, proteins which inhibit PLA2 have been found to be common to tissues containing PLA2 activity (Hostetier et al 1982;DiRosa et al 1988;Kunze et al 1988), and the same may be true for boar spermatozoa.…”