Hansford R G
J Biol Chem. 1977 Mar 10;252(5):1552-60.
The oxidation of an optimal concentration of palmitoyl-carnitine, buffered with bovine serum albumin, by isolated rat heart mitochondria was found to give rise to an inactivation of pyruvate dehydrogenase, provided that the concentration of pyruvate present in the mitochondrial incubation was less than 250 muM. The greatest degree of inactivation was found at the lowest pyruvate concentration used, 50 muM, and this concentration was adopted for further studies in which the rate of mitochondrial respiration was varied. This was done by varying the activity of added hexokinase, in the presence of ATP, MgCl2, and glucose, and thus the availability of ADP to the mitochondrion. The pyruvate concentration in the incubation was approximately stabilized by adding pyruvate on the basis of oxygen consumption, with the ratio of pyruvate consumed:O2 consumed determined by trial and error. This device allowed the maintenance of essentially steady pyruvate concentrations and ATP/ADP ratios for at least 5 min, and allowed the pyruvate dehydrogenase interconversion time to approach a steady state. Activities of pyruvate dehydrogenase after 5 or 6 min of respiration were as follows, with values given in nanomoles/min/mg of protein for incubations containing pyruvate as sole substrate, and values for incubations containing pyruvate plus palmitoylcarnitine given in parentheses: State 4, 27 (9); 55% of State 3, 54 (14); 85% of State 3, 73 (28); State 3, 90 (93). Respiratory states are defined by Chance and Williams (1955) J. Biol. Chem. 217, 409-427). Values at earlier time points are also presented so that some idea may be formed of the time course of pyruvate dehydrogenase inactivation. CoASH/acetyl-CoA, NAD+/NADH, and ATP/ADP ratios were measured at the same time points in precisely scaled up incubations. The presence of palmitoylcarnitine in State 4 was found to give essentially no change in NAD+/NADH and ATP/ADP ratios and thus the inactivation of pyruvate dehydrogenase in that state may be attributed to a decreased CoASH/acetyl-CoA ratio. At a respiratory rate of 85% of State 3, palmitoylcarnitine did not change the ATP/ADP ratio, but lowered both CoASH/acetyl-CoA and NAD+/NADH ratios, both of which may contribute to pyruvate dehydrogenase inactivation. In State 3 there was no pyruvate dehydrogenase inactivation, despite a lowered CoASH/acetyl-CoA ratio in the presence of palmitoylcarnitine. It is concluded that ATP/ADP ratio has a pronounced effect on the interconversion of active and inactive pyruvate dehydrogenase, in according with previous work. Moreover, at a given ATP/ADP ratio, the effects of palmitoylcarnitine oxidation on enzyme interconversion are consistent with a mechanism involving the modulation of the interconversion by NAD+/NADH and CoASH/acetyl-CoA ratios...
研究发现，用牛血清白蛋白缓冲的最佳浓度的棕榈酰肉碱，在离体大鼠心脏线粒体中被氧化时，会导致丙酮酸脱氢酶失活，前提是线粒体孵育体系中丙酮酸的浓度低于250 μM。在所用的最低丙酮酸浓度（50 μM）下观察到最大程度的失活，后续进一步研究中均采用该浓度，在此研究中改变线粒体呼吸速率。这通过在ATP、MgCl₂和葡萄糖存在的情况下改变添加的己糖激酶的活性来实现，从而改变线粒体可利用的ADP量。孵育体系中的丙酮酸浓度通过根据耗氧量添加丙酮酸大致稳定下来，丙酮酸消耗与O₂消耗的比例通过反复试验确定。该方法可使丙酮酸浓度和ATP/ADP比例在至少5分钟内基本保持稳定，并使丙酮酸脱氢酶的相互转化时间接近稳态。呼吸5或6分钟后丙酮酸脱氢酶的活性如下，以纳摩尔/分钟/毫克蛋白质为单位，仅含丙酮酸作为底物的孵育体系的值列于括号内，含丙酮酸加棕榈酰肉碱的孵育体系的值列于括号外：状态4，27（9）；状态3的55%，54（14）；状态3的85%，约73（28）；状态3，90（93）。呼吸状态由Chance和Williams（1955年，《生物化学杂志》217卷，409 - 427页）定义。还给出了更早时间点的值，以便对丙酮酸脱氢酶失活的时间进程有大致了解。在精确放大的孵育体系中，于相同时间点测量了CoASH/乙酰辅酶A、NAD⁺/NADH和ATP/ADP比例。发现在状态4下存在棕榈酰肉碱时，NAD⁺/NADH和ATP/ADP比例基本不变，因此该状态下丙酮酸脱氢酶的失活可能归因于CoASH/乙酰辅酶A比例降低。在呼吸速率为状态3的85%时，棕榈酰肉碱不改变ATP/ADP比例，但降低了CoASH/乙酰辅酶A和NAD⁺/NADH比例，这两者都可能导致丙酮酸脱氢酶失活。在状态3下，尽管存在棕榈酰肉碱时CoASH/乙酰辅酶A比例降低，但丙酮酸脱氢酶没有失活。结论是，与之前的研究一致，ATP/ADP比例对活性和非活性丙酮酸脱氢酶的相互转化有显著影响。此外，在给定的ATP/ADP比例下，棕榈酰肉碱氧化对酶相互转化的影响与一种涉及由NAD⁺/NADH和CoASH/乙酰辅酶A比例调节相互转化的机制相符……
