Babcock D F, First N L, Lardy H A
J Biol Chem. 1975 Aug 25;250(16):6488-95.
Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both adenosine and guanosine di- and triphosphates were labeled, but contrary to expectations, the specific activity of ADP exceeded that of ATP. The uptake of phosphate and its incorporation into nucleotides were suppressed by respiratory inhibitors and were abolished by treatment with sulfhydryl-directed reagents at 10 to 20 nmol/mg of sperm protein. With fructose as an energy source for motility, glycolysis did not support phosphate uptake. Nucleotide labeling was increased 60 to 80-fold when the cells were treated with the polyene antibiotic filipin, and filipin was able to reverse the inhibition of phosphate (and succinate) entry produced by N-ethylmaleimide or mersalyl. Since filipin interacts specifically with the cholesterol-containing plasma membrane of bovine spermatozoa and increases its permeability, it is probable that the plasma membrane normally limits phosphate and succinate transport into these cells. This contention is further supported by the observation that high concentrations of extracellular Pi, the penetration of which was extremely limited under these conditions, protected against inactivation by N-ethylmaleimide. Phosphate uptake was increased 10 to 20-fold, but nucleotide labeling was inhibited, when calcium was present in the incubation medium. Ruthenium red, presumably acting extracellularly, prevented these effects of calcium. Thus, the entry of phosphate and succinate into spermatozoa is controlled by plasma membrane components that resemble the phosphate and succinate exchangers and calcium carrier found in mitochondria isolated from other sources.
牛精子在体外有氧孵育期间积累了少量的³²Pi。至少50%获得的同位素迅速进入细胞核苷酸。腺苷和鸟苷的二磷酸和三磷酸都被标记，但与预期相反，ADP的比活性超过了ATP。磷酸盐的摄取及其掺入核苷酸的过程受到呼吸抑制剂的抑制，并且在用巯基导向试剂以10至20 nmol/mg精子蛋白处理时被消除。以果糖作为运动的能量来源时，糖酵解不支持磷酸盐的摄取。当细胞用多烯抗生素制霉菌素处理时，核苷酸标记增加了60至80倍，并且制霉菌素能够逆转由N-乙基马来酰胺或汞撒利产生的对磷酸盐（和琥珀酸盐）进入的抑制作用。由于制霉菌素与牛精子含胆固醇的质膜特异性相互作用并增加其通透性，质膜很可能通常限制磷酸盐和琥珀酸盐向这些细胞内的转运。高浓度的细胞外Pi（在这些条件下其渗透极其有限）能防止N-乙基马来酰胺导致的失活，这一观察结果进一步支持了这一论点。当孵育培养基中存在钙时，磷酸盐摄取增加了10至20倍，但核苷酸标记受到抑制。钌红大概在细胞外起作用，阻止了钙的这些效应。因此，磷酸盐和琥珀酸盐进入精子是由类似于从其他来源分离的线粒体中发现的磷酸盐和琥珀酸盐交换体以及钙载体的质膜成分控制的。
