Studies on the gene expression and the physiological functions of nucleotide pyrophosphatase

1995 Fiscal Year Final Research Report Summary

• Project/Area Number: 06672212
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Biological pharmacy
• Research Institution: Kyoto Sangyo University
• Principal Investigator: FUNAKOSHI Ikuo Kyoto Sangyo Univ., Fac.of Engineering, Professor, 工学部, 教授 (10025702)
• Project Period (FY): 1994 – 1995
• Keywords: nucleotide pyrophosphatase / gene expression / initiation AUG / tyrosine kinase inhibitor

Research Abstract

1. There are two AUG codons within the same open reading frame on the 5'-terminal region of human NPPase mRNA.In order to determine which AUG is the real initiation codon for NPPase, we constructed expression vectors carrying a long form cDNA,which contained the two AUG,or a short form cDNA,which contained only the downstream AUG.When the constructs were introduced into CHO cells, expression of NPPase was observed in the CHO cells transfected with the long form construct but not with the short form construct. We also constructed mutant cDNAs in which either of the two AUG codons converted to codons not to be able recognize as a initiation codon by mammalian translation system, and then the ability of each mutant to produce NPPase was assessed by in vitro transcription/translation system. In this experiment, the mutant at the upstream AUG could not direct the synthesis of NPPase. These results shows that the translation of human NPPase mRNA is initiates at the upstream AUG.
2. Insulin resistant cells, such as fibroblasts from patients with non-insulin dependent diabates or MDA-MB-231 human breast cancer cell, contain tyrosine kinase inhibitory activity. Goldfine et al. suggested that the inhibitor may be NPPase. To address this issue, we compared the NPPase activity, the amount of NPPase and the amount of NPPase mRNA in human breast cancer cells, MDA-MB-231. with those in insulin sensitive breast cancer cells, ZR-75-1. Each measurement showed very higher value in MDA-MB-231 than in ZR-75-1. We also studied whether the cell extract of the fibroblasts from patients with Lowe's syndrome which contain larger amount of NPPase inhibit insulin receptor tyrosine kinase activity in ZR-75-1 cell. The kinase activity was markedly inhibited by the cell extract from NC cells. Therefore it is possible that insulin receptor tyrosine kinase in some insulin resistant cell is inhibited by NPPase.