Regulation Mechanism of Glycolysis in Skeletal Muscle

为阐明骨骼肌糖酵解的调控机制，研究了6-磷酸果糖2-激酶/2，6-二磷酸果糖酶(F6P2K)的组织特异性表达模式。F6P2K催化糖酵解最有效的激活剂2，6-二磷酸果糖(F26P)的合成和降解。这些酶活性的比例决定了细胞内F26P的含量。已在肝脏、心脏、睾丸和脑中鉴定出该酶的四个组织特异性同工酶基因。最近，我们发现这些基因以组织特异性的方式通过选择性剪接至少产生15个mRNAs。该酶的变化对于糖酵解的组织特异性调节非常重要。在本研究中，我们证明了9个mRNAs的存在，即RM2K、RH2 K1、RH2 K4、RB2K2、RB2K3、RB2K5、RB2K6、RB2K7和RB2K8。已知RM2K在骨骼肌中以蛋白质的形式表达。然而，异构体不能调节糖酵解，因为它催化降解而不是合成。因此，我们以寡肽为抗原制备抗体，用免疫印迹法检测各亚型。令人惊讶的是，在骨骼肌中没有检测到异构体。有趣的是，虽然RB2K7和RB2K8在脑、骨骼肌和心脏中的表达水平相同，但RB2K7亚型仅在心脏中检测到。这些结果表明，RB2K7和RB2K8蛋白的表达受翻译和/或翻译后过程的调节，但不受其mRNA水平的调节。

Tissue-specific expression pattern of fructose-6-phosphate 2-kinase/fructose 2,6-bisphosphatase (F6P2K) was studied to elucidate a regulation mechanism of glycolysis in skeletal muscle. The F6P2K catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (F26P) which is the most potent activator of glycolysis. The ratio of these activities of the enzyme determines the intracellular content of F26P. Four tissue-specific isozyme genes of the enzyme have been identified in liver, heart, testis and brain. Recently, we have found that the genes produce at least fifteen mRNAs by alternative splicing in a tissue-specific manner. The variety of the enzyme is very important for tissue-specific regulation of glycolysis. In this study we demonstrate the occurrence of nine mRNAs, namely, RM2K, RH2K1, RH2K4, RB2K2, RB2K3, RB2K5, RB2K6, RB2K7 and RB2K8. It is already known that the RM2K is expressed as a protein in skeletal muscle. However, the isoform cannot regulate the glycolysis because it catalyzes the degradation but not the synthesis. Therefore, we prepared antibodies using oligopeptides as antigens to detect each isoform by western blot. Surprisingly, no isoform was detected in skeletal muscle. Interestingly, the RB2K7 isoform was detected only in heart, although the RB2K7 and the RB2K8 mRNAs were expressed in brain, skeletal muscle and heart at the same level. These results indicate that the expression of the RB2K7 and RB2K8 proteins is regulated by translational and/or post-translational processes but not by their mRNA levels.