Leucine degradation in cell-free extracts of skeletal muscle.
Leucine degradation in cell-free extracts of skeletal muscle.
复制标题
骨骼肌无细胞提取物中亮氨酸的降解。
作者：
R. Odessey;A. Goldberg
由于骨骼肌是体内亮氨酸、异亮氨酸和缬氨酸氧化的主要部位，因此在大鼠肌肉的无细胞制备物中研究了亮氨酸氧化的途径和控制。亮氨酸被发现转氨为4-甲基-2-氧代戊酸，然后氧化脱羧。在差速离心中，70- 80%的转氨酶活性在细胞的可溶性部分中回收，剩余量在线粒体部分中回收。两种组分的转氨酶具有相似的最适pH值，并且都被Ca ~（2+）显著抑制。因此，细胞Ca 2+浓度的变化可能会调节转氨酶活性。这两种转氨酶对2-酮戊二酸的亲和力比丙酮酸高得多。因此，在体内利用亮氨酸的氨基在肌肉中生物合成丙氨酸[Odessey，Khairallah & Goldberg（1974）J.Biol.Chem.249，7623- 7629]涉及用2-酮戊二酸转氨基以产生谷氨酸，然后用丙酮酸转氨基以产生丙氨酸。通过甲基-2-氧代[1- 14 C]戊酸脱羧测定的脱氢酶活性仅位于含有线粒体的级分中，并且需要NAD+、CoA和焦磷酸硫胺素以获得最佳活性。竞争性抑制的测量表明，亮氨酸，异亮氨酸和缬氨酸的含氧酸都是由相同的酶脱羧。酶的活性下降了90%，冷冻或超声处理后，并刺激几倍的Mg 2+，K+和磷酸根离子。此外，它是显着抑制ATP，但不被非代谢类似物。这一观察结果表明，ATP的分裂是抑制所必需的。脱氢酶对4-甲基-2-氧代戊酸的氧化脱羧似乎是肌肉匀浆和完整组织中亮氨酸氧化的限速步骤。事实上，与[1- 14 C]亮氨酸孵育的大鼠肌肉以与脱羧速率相当的速率向培养基中释放1- 14 C标记的含氧酸。完整的肌肉也释放[1- 14 C]缬氨酸或[1- 14 C]异亮氨酸的含氧酸，但不释放其他氨基酸。这些发现表明，肌肉是血液中支链含氧酸的主要来源。
Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.