Effects of nucleotides on N-acetyl-d-glucosamine 2-epimerases (renin-binding proteins): comparative biochemical studies.
Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GlcNAc 2-epimerase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GlcNAc 2-epimerase activity. Other nucleotides had no effect on human GlcNAc 2-epimerase activity. Rat and porcine GlcNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GlcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.
Takahashi
S
S
Department of Bioengineering, Akita Research Institute of Food and Brewing (ARIF), 4-26 Sanuki, Arayamachi Akita 010-1623, Japan. saori@arif.pref.akita.jp
Hori
K
K
Takahashi
K
K
Ogasawara
H
H
Tomatsu
M
M
Saito
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002238:Carbohydrate Epimerases / Q000187:drug effects* / Q000302:isolation & purification / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects* / Q000302:isolation & purification / Q000494:pharmacology; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D009711:Nucleotides / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects* / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine; D013820:Thermolysin / Q000037:antagonists & inhibitors*; D000092502:Renin Inhibitors
D000255:Adenosine Triphosphate; D002238:Carbohydrate Epimerases; D002352:Carrier Proteins; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; D011994:Recombinant Proteins; D013820:Thermolysin; D000092502:Renin Inhibitors; C029603:deoxyguanosine triphosphate; C024519:N-acyl-D-glucosamine 2-epimerase; C498259:RENBP protein, human; C498258:Renbp protein, rat; C026600:2'-deoxyadenosine triphosphate