Van Dop C, Hutson S M, Lardy H A
J Biol Chem. 1977 Feb 25;252(4):1303-8.
Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.
用鲁塔霉素或鱼藤酮处理牛附睾精子，会抑制内源性底物支持的呼吸作用和运动能力。当用各种抑制剂阻断氧化磷酸化时，丙酮酸会被代谢以产生ATP并恢复运动能力。通过糖酵解代谢产生ATP的果糖，也能够使细胞复苏。测试的其他底物（乳酸、乙酸、α-酮戊二酸或乙醛酸）无法恢复经鲁塔霉素处理的细胞的运动能力。在丙酮酸存在的情况下，磷酸化解偶联剂羰基氰化物-对-三氟甲氧基苯腙会降低未处理细胞或经鲁塔霉素或鱼藤酮处理的细胞的运动能力和ATP水平至正常水平。因此，丙酮酸通过一条独立于与电子传递链相关的氧化磷酸化的途径被代谢以产生ATP。硫辛酰胺脱氢酶抑制剂5-甲氧基吲哚-2-羧酸可阻止经鲁塔霉素处理的细胞中运动能力和ATP水平的升高，这表明当存在鲁塔霉素时，α-酮酸氧化参与了丙酮酸产生ATP的过程。在丙酮酸存在的情况下，邦克里酸、抗霉素A和无氧环境会消除运动能力，将ATP降低至低水平，并且还会显著降低丙酮酸的代谢速率。只有当细胞ATP浓度较低时，丙酮酸才会产生乙酸。游离肉碱浓度的降低表明最初使用的丙酮酸会转化为乙酰肉碱。结果表明，精子特有的线粒体内乳酸脱氢酶X允许丙酮酸氧化产生的NADH将其他丙酮酸分子还原为乳酸。因此，丙酮酸与电子传递链的NADH脱氢酶竞争，并可替代它。在各种条件下，丙酮酸都能迅速补充乙酰肉碱库。
