Study on metabolic control function of adenylate kinase

腺苷酸激酶有助于细胞腺嘌呤核苷酸组成的稳态。已鉴定出三种同工酶：胞浆型ak1，线粒体膜间型AK2和线粒体基质型ak3。然而，这种不同的亚细胞分布的生理意义仍有待解决。本研究旨在分析引入和表达腺苷酸激酶同工酶cdna后，酶的亚细胞分布变化对细胞代谢的影响。为了控制酶的亚细胞分布，了解细胞室特别是线粒体的靶向信号是必不可少的。本研究证实AK3分子不像其他线粒体蛋白那样具有可切割的n端序列。当AK3 cDNA在大肠杆菌中表达时，AK3蛋白通过细菌内膜被输出到质周间隙。构建了AK3 n端（残基6 - 23）与β -内酰胺酶融合蛋白的表达质粒。当该质粒在大肠杆菌中表达时，融合蛋白在周质中被回收。这些结果表明AK3的n端序列参与了对线粒体基质的靶向。

Adenylate kinase contributes to homeostasis of the cellular adenine nucleotide composition. Three isozymes have been identified: the cytosol type-AK1, the mitochondrial intermembraneous-type AK2, and the mitochondrial matrix type-AK3. However, the physiological significance of such different subcellular distribution remains to be solved.This study aims at the analysis of the effect of changes in the subcellular distribution of the enzymes on the cellular metabolism after introduction and expression of cDNAs for adenylate kinase isozymes. To control the subcellular distribution of the enzymes, knowledge on the targeting signal to cellular compartments especially to mitochondria is essential. This study established that the AK3 molecule has no cleavable N-terminal sequence as found in other mitochondrial proteins. When the AK3 cDNA was expressed in Escherichia coli, the AK3 protein was exported to the periplasmic space through the bacterial inner membrane. An expression plasmid was constructed which directs a fusion protein consisting of the N-terminal portion of AK3 (residues 6 - 23) and beta- lactamase. When this plasmid was expressed in E. coli, the fusion protein was recovered in the periplasm. These results indicate that the involvement of the N-terminal sequence of AK3 in targeting to the mitochondrial matrix.