McKenna M C, Tildon J T, Couto R, Stevenson J H, Caprio F J
Department of Pediatrics, University of Maryland School of Medicine Baltimore 21201.
Neurochem Res. 1990 Dec;15(12):1211-20. doi: 10.1007/BF01208582.
Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-[U-14C]malate in primary cultures of rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from 0.01 mM and 0.5 mM malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain. Both the biphasic kinetics and the differential action of many of the effectors on the 14CO2 production from 0.01 mM and 0.5 mM malate provide evidence for the presence of more than one pool of malate metabolism in cultured rat brain astrocytes.
由于已知苹果酸在大脑的多种功能中发挥重要作用，包括能量代谢、还原当量的转移以及不同细胞类型之间可能的代谢物质运输；因此开展了一系列生化测定，以评估大鼠脑星形胶质细胞原代培养物中L-[U-¹⁴C]苹果酸产生¹⁴CO₂的速率。代谢抑制剂鱼藤酮和抗霉素A几乎完全抑制了标记苹果酸产生¹⁴CO₂，这表明大部分苹果酸代谢与电子传递系统相关。对标记苹果酸代谢产生¹⁴CO₂的双倒数作图显示出双相动力学，具有两个明显的Km和Vmax值，表明这些细胞中存在不止一种苹果酸代谢机制。随后使用0.01 mM和0.5 mM苹果酸进行实验，以确定添加效应剂是否会不同程度地改变高浓度和低浓度苹果酸的代谢。所研究的效应剂包括可能是苹果酸代谢内源性调节剂的化合物以及能提供有关调节苹果酸代谢机制信息的代谢抑制剂。乳酸和天冬氨酸同等程度地降低了0.01 mM和0.5 mM苹果酸产生¹⁴CO₂的量。然而，鉴定出了一些效应剂，它们选择性地改变了0.01 mM苹果酸的代谢，包括氨基氧乙酸、速尿、N-乙酰天冬氨酸、草酰乙酸、丙酮酸和葡萄糖，但对0.5 mM苹果酸的代谢几乎没有影响。此外，α-酮戊二酸和琥珀酸降低0.01 mM苹果酸产生¹⁴CO₂的量比降低0.5 mM苹果酸产生¹⁴CO₂的量更多。相反，一些效应剂对0.5 mM苹果酸代谢的改变比对0.01 mM苹果酸代谢的改变更大。这些效应剂包括蛋氨酸亚砜亚胺、谷氨酸、丙二酸、α-氰基-4-羟基肉桂酸和哇巴因。双相动力学以及许多效应剂对0.01 mM和0.5 mM苹果酸产生¹⁴CO₂的差异作用均为培养的大鼠脑星形胶质细胞中存在不止一个苹果酸代谢池提供了证据。
