| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
In vertebrates, two isozymes of adenylate kinase, the musclr type (AK1) and the liver type (AK2), have been identified, each of which has distinct distribution in tissues as well cellular compartment. Though AK2 is localized in the mitochondrial intermembraneous space. cDNA sequence analysis revealed that no cleavable presequence is present in its NH_2-terminal region as found in the imported mitochondrial proteins. To know the sorting sequence of the protein to the mitochondrial compartment, we examined the in vitro binding study of AK2 protein. The AK2 cDNA inserted downstream from the SP6 promoter served as a template for RNA synthesis by the in vitro transcription system. AK2 mRNA was isolated and used as a template of in vitro translation system of the rabbit reticulocyre lysate. After incubating the synthesized protein with purified rat mitochondria, more than 50% of the protein bound to the mitochondria. In contrast, we could not demonstrate the binding of AK1 protein by the same analysis. Thus, it was proved that AK2 protein itself has the binding activity to mitochondria.
Gene organizations of human and chicken AK1 and bovine AK2 were determined. Both of the AK1 genes were divided into 7 exons and the positions of introns were exactly the same in these two genes. When the amino acid sequence of chicken AK1 was compared with that of bovine AK2, 41% of the residues were homologous. There was a gap of about 30-amino acid residues between exon 5 and 6, which corresponded to one exon in AK2 gene. Taking into account of the close relationship of AK2 to yeast and E. coli adenylate kinases, it is considered that AK1 gene was derived from AK2 gene by deletion of the exon.
Thus, adenylate kinase probably changed its ability to localize in the cytosol of skeletal muscle, brain and erthrocyte to fulfill the specialized function of their organs and tissues through the moleculer evolution of AK2.
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