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A B S T RA C T This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na,K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [y-s*p]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37°C, but not at 0°C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [7-a*P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as 32Pi liberated during the subsequent incubation at 0°C, reflects completely the differential effects of preincubation with ATP on 3,p incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na,K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na,K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in seminal fluid, as well as levels of sperm lipid membrane peroxidation, were investigated in fertile and infertile men. Semen samples, obtained by masturbation from 37 infertile and 14 fertile men, were examined for the presence of TNF-alpha and IL-6. The level of lipid peroxidation of the sperm membrane was measured by determining malondialdehyde (MDA) formation. The correlation between the IL-6 and the TNF-alpha concentrations in seminal plasma with the levels of lipid peroxidation of the sperm membranes was statistically evaluated. The IL-6 concentration in seminal plasma of infertile men was significantly higher than that of fertile men (p < .05). Similarly, the level of membrane lipid peroxidation was higher for the semen of infertile men than that of fertile men (p < .001). A significant positive correlation was found between IL-6 levels in seminal plasma and membrane sperm lipid peroxidation (p < .002), but not between this parameter and TNF-alpha levels in seminal plasma. These findings suggest a possible association between IL-6 seminal plasma levels and lipid peroxidation of sperm membrane. Stimulation of reactive species production by human sperm and leucocytes, induced by the high levels of IL-6, could explain these results.

Several pro-inflammatory cytokines at physiological concentrations increase the level of lipid peroxidation of sperm membranes, which could be important for the sperm fecundation process. However, infection-inflammation concentrations of some cytokines, such as IL-8 and TNF-alpha, either alone or in the presence of leukocytes, could drive the lipid peroxidation of the spermatozoa plasma membrane to levels that can affect the sperm fertility capacity.

The phosphoproteins formed by incubation of red cell ghosts with [y-8 2 P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The 2p_ labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of 3 2 P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the 3sP-phosphoprotein present in the gels. The molecular weight of the 2P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na: K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.