Hutson S M, Van Dop C, Lardy H A
J Biol Chem. 1977 Feb 25;252(4):1309-15.
Treatment with the polyene antibiotic, filipin, renders the spermatozoan cell membrane permeable to small molecules, but not to the intracellular enzymes aldolase and lactate dehydrogenase. Pyruvate (10 mM) as the sole substrate was metabolized very slowly. L-Carnitine increased pyruvate metabolism 3- to 4-fold and allowed limited rates of oxidative phosphorylation. When spermatozoa treated with filipin were supplemented with malate, there was a rapid, almost linear rate of pyruvate metabolism which was slightly increased by L-carnitine. In the absence of malate, 20 to 30% of the pyruvate used was reduced to lactate; this increased to 57% in the presence of malate. Without malate, about 90% of the pyruvate metabolized was converted to lactate and acetate or L-acetylcarnitine. Rutamycin or rotenone increased both the rate of pyruvate use and the delta lactate/deltapyruvate ratio. Under all treatments, L-carnitine consistently reduced the percentage of pyruvate converted to lactate by about 10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine rather than lactate. The mitochondrial inhibitors, rotenone or rutamycin, did not change the amount of pyruvate that was converted to metabolites other than lactate, or L-acetylcarnitine, or both. Pyruvate-supported State 3 respiration was linear only if L-carnitine, or malate, or both, were added to the incubation medium. Added malate was necessary to produce a rapid State 3 respiratory rate and was also required for significant respiratory activity in the presence of rotenone or rutamycin. From cells metabolizing [2-14C]pyruvate (1.4 mM), 14C-labeled acid-extractable metabolites were separated by ion exchange column chromatography. All of the [2-14C]pyruvate (+/-5%) used was recovered in 14C-labeled metabolites and 14CO2. In the presence of malate, citrate accumulation was significant, and was always large in comparison to flux through the citric acid cycle. Glutamate, beta-hydroxybutyrate, acetoacetate, fumarate, aspartate, and alpha-ketoglutarate did not accumulate in significant amounts. Some 14C-labeled succinate was produced but only in the presence of malate. Alkaline hydrolysis of a fraction containing carnitine esters yielded acetate and a compound tentatively identified as beta-hydroxybutyrate or lactate. As in intact cells, intramitochondrial lactate dehydrogenase competes successfully with the electron transport system for the NADH generated by pyruvate metabolism. The role of lactate and L-carnitine, and conclusions suggested by the accumulation of certain metabolites are discussed in relation to control of citric acid cycle activity.
用多烯抗生素制霉菌素处理后，精子细胞膜对小分子具有通透性，但对细胞内的醛缩酶和乳酸脱氢酶则不然。丙酮酸（10 mM）作为唯一底物时代谢非常缓慢。左旋肉碱使丙酮酸代谢增加3至4倍，并使氧化磷酸化速率有限。当用制霉菌素处理的精子补充苹果酸时，丙酮酸代谢迅速，几乎呈线性速率，左旋肉碱使其略有增加。在没有苹果酸的情况下，所消耗的丙酮酸中有20%至30%被还原为乳酸；在有苹果酸的情况下，这一比例增加到57%。没有苹果酸时，代谢的丙酮酸中约90%转化为乳酸、乙酸或L-乙酰肉碱。鲁塔霉素或鱼藤酮增加了丙酮酸的利用速率以及乳酸/丙酮酸的比值。在所有处理条件下，左旋肉碱始终使转化为乳酸的丙酮酸百分比降低约10%；部分丙酮酸优先分流到L-乙酰肉碱而不是乳酸中。线粒体抑制剂鱼藤酮或鲁塔霉素并没有改变转化为除乳酸、L-乙酰肉碱或两者之外的其他代谢物的丙酮酸量。只有在向孵育培养基中添加左旋肉碱、苹果酸或两者时，丙酮酸支持的状态3呼吸才呈线性。添加苹果酸对于产生快速的状态3呼吸速率是必要的，并且在存在鱼藤酮或鲁塔霉素时对于显著的呼吸活性也是必需的。从代谢[2-14C]丙酮酸（1.4 mM）的细胞中，通过离子交换柱色谱法分离出14C标记的酸可提取物代谢物。所使用的所有[2-14C]丙酮酸（±5%）都在14C标记的代谢物和14CO2中回收。在有苹果酸的情况下，柠檬酸积累显著，并且与通过柠檬酸循环的通量相比总是很大。谷氨酸、β-羟基丁酸、乙酰乙酸盐、富马酸盐、天冬氨酸和α-酮戊二酸没有大量积累。仅在有苹果酸的情况下产生了一些14C标记的琥珀酸盐。对含有肉碱酯的一部分进行碱性水解，产生乙酸盐和一种初步鉴定为β-羟基丁酸或乳酸的化合物。与完整细胞一样，线粒体内的乳酸脱氢酶成功地与电子传递系统竞争丙酮酸代谢产生的NADH。结合柠檬酸循环活性的控制，讨论了乳酸和左旋肉碱的作用以及某些代谢物积累所暗示的结论。
