Depression of human sperm motility by inhibition of enzymatic methylation

Alteration of membrane fluidity during enzymatic methylation of membrane phosphatidyl-ethanolamine (PE) and neutralization of negative charges of membrane proteins due to methylation of carboxyl groups may contribute to sperm motility. Therefore, enzymatic phospholipid methylation and carboxymethylation, and the consequences of their inhibition on motility, were studied using human sperm. These studies gave the following results. Human sperm homoganates contained two phospholipid N-methyltransferases (PMT) which converted PE to phosphatidylcholine (PC) in the presence of S-adenosylmethionine (SAM). The first PMT converted PE to phosphatidyl-N-methylethanolamine (PME). In had a Km of 4.0 microM and a pH optimum of 8.0. The second PMT converted PME to phosphatidyl-N,N-dimethylethanolamine and PC. It had a Km of 71 microM and a pH optimum of 10.0. Spermatozoa also contained protein carboxymethylase (PCM) and methyl aceptor protein (MAP). The intracellular levels of S-adenosylhomocysteine (SAH), an inhibitor of SAM-mediated methylations, were increased by adding adenosine (100 microM), L-homocysteine thiolactone (L-HCT, 10 microM), and erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 microM), an inhibitor of adenosine deaminase, to human sperm ejaculates that had been diluted with sodium phosphate buffer at pH 7.4 and 25 degrees. The motility index of each sperm suspension was determined every hour for 4 hr. In the presence of the mixture of adenosine, L-HCT and EHNA, the motility index was depressed by 57%. Under similar conditions, phospholipid methylation was depressed by 48%. Similar experiments were also conducted in the presence of 3-deazaadenosine (Deaza, 80 microM), a selective inhibitor of SAH hydrolase. In the presence of adenosine and L-HCT, Deaza depressed the motility index by 60% and phospholipid methylation by 86%. The potencies of SAH in the inhibition of phospholipid methylation and protein carboxymethylation in sperm homogenates had the following order: PMT I greater than PCM greater than PMT II. These observations indicate that the PMT system and/or the PCM-MAP system play a significant role in the regulation of human sperm motility.     

Action of mercurials on 3H-cAMP binding to the regulatory subunit-II of cAMP-dependent protein kinase.

Binding of the regulatory subunit type II (RII) of adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase was inhibited by Hg2+ with an IC50 value of 0.31 microM. Methyl mercury, p-chloromercuribenzoic acid (PCMB), and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) also inhibited cAMP binding with IC50 values of 70-80 microM for organic mercurials and 130 microM for DTNB. Addition of 1 mM 2-mercaptoethanol and 1 M cysteine to the assay mixture reversed these inhibitions. N-ethylmaleimide (NEM) showed little effect on the binding. On the other hand, Hg2+ and methyl mercury markedly suppressed enzymatic activity of the catalytic subunit. The IC50 value was 0.13 microM for Hg2+ and 0.15 microM for organic mercurials. Scatchard plots of kinetic analysis data for the cAMP binding revealed a noncompetitive type of inhibition by mercurials and DTNB. It is suggested that blockade of sulfhydryl groups resulted in the inhibition of cAMP binding to the RII subunit, which might result in preserving the association of the RII subunit and the catalytic subunits and in preventing further inactivation of the catalytic subunit by Hg2+.     

Inhibition by reactive aldehydes of superoxide anion radical production from stimulated polymorphonuclear leukocytes and pulmonary alveolar macrophages. Effects on cellular sulfhydryl groups and NADPH oxidase activity

Alpha,beta-unsaturated aldehydes such as acrolein (ACR) and crotonaldehyde (CRO) have been shown previously in our laboratory to inhibit the production of superoxide anion radical (O2-) by stimulated phagocytic cells in vitro in a dose-related manner. Based on the known reactivity of these compounds towards cellular sulfhydryls (SH), the present studies were aimed at investigating cellular SH status in relation to O2- production. Plasma membrane surface SH groups were measured using carboxypyridinedisulfide and monitoring the resultant formation of mixed disulfides through assay of thione released into the supernatant fraction. Intracellular non-protein sulfhydryls were measured using 5,5'-dithiobis-2-nitrobenzoic acid. In both human polymorphonuclear leukocytes (PMN) and rat pulmonary alveolar macrophages (PAM) there was a dose-related decrease in surface SH and soluble SH after ACR and CRO treatment. Propionaldehyde, a three-carbon saturated aldehyde, was without effect. The decrease in surface SH was greater than the decrease in soluble SH. In addition, in PMN and PAM preincubated with 5-40 microM ACR, there was a dose-related inhibition in the rate of O2- production with no effect on the lag time as measured by cytochrome c reduction. In stimulated PMN, there was a dose-related decrease in the rate after addition of 5-40 microM ACR. These data suggest that changes in SH status by reactive aldehydes can modulate the activity of the plasma membrane NADPH oxidase responsible for O2- production.