Analysis of multi-function of renin binding protein

点击翻译按钮获取中文摘要

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.