| Project/Area Number |
16580082
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Akita Research Institute of Food and Brewing |
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Principal Investigator |
TAKAHASHI Saori Akita Research Institute of Food and Brewing, Department of Bioengineering, Head of Department, 総合食品研究所・生物機能部門, 主席研究員 (10142184)
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| Co-Investigator(Kenkyū-buntansha) |
HORI Kazuyuki Akita Research Institute of Food and Brewing, Department of Bioengineering, Senior Researcher, 総合食品研究所, 主任研究員 (50181516)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | renin / binding protein / nucleotides / ATP / N-acetyl-D-mannosamine / epimerase / multi-functional protein / 多機能タンパク質 / 部位特異的変異体 / 高血圧 |
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| Research Abstract |
Renin binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. Recently the protein was identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The enzyme catalyzes the interconversion between GlcNAc and N-acetyl-D-mannosamine (ManNAc) and nucleotide are essential as a cofactor. Our recent studies demonstrated that the middle domain of GlcNAc 2-epimerase participates in the specificity for and binding of nucleotide. To identify the residue conferring nucleotide binding, amino acid substitutions were introduced in the human and rat GlcNAc 2-epimerases. The expression and characterization of the mutants indicate that reside 171 of GlcNAc 2-epimerase is critical for the nucleotide binding of the enzyme. The recombinant porcine RnBP inhibited porcine renin activity in a dose dependent manner in the absence of nucleotide. But the inhibition was neutralized by nucleotides such as ATP,dATP,dGTP,dCTP, or dTTP. ATP inhibited the formation of hetero-complex of renin with RnBP. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited GlcNAc 2-epimerase activity concomitant decaying dimer to monomer of the enzyme. NEM did not inhibit GlcNAc 2-epimerase activity in the presence of ATP. These results clearly indicate that nucleotides stabilize dimeric from of RnBP (GlcNAc 2-epimerase) and inhibited the formation of renin-RnBP hetero-complex, HMW renin. Moreover, the human renin was expressed in E. coli cells as a fusion protein of thioredoxin. The chimeric protein, accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by L-arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the renin activity. The active recombinant human renin will be used for the physiological studies of human RnBP in near future.
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