Oxidative refolding of bovine pancreatic RNases A and B promoted by Asn-glycans.
It was previously revealed [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477] that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions. To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions. The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages. In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates. Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the high-mannose type. In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.
Nishimura
I
I
Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka 593-8231, Japan.
Uchida
M
M
Inohana
Y
Y
Setoh
K
K
Daba
K
K
Nishimura
S
S
Yamaguchi
H
H
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000494:pharmacology; D002417:Cattle; D005453:Fluorescence; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000378:metabolism* / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine
D000817:Anilino Naphthalenesulfonates; D002241:Carbohydrates; D011134:Polysaccharides; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic