Purification and characterization of a human urine ribonuclease (RNAase 1) showing genetic polymorphism.
A ribonuclease (RNAase) was isolated and purified from the urine of a 45-year-old man by column chromatographies on DEAE-Sepharose CL-6B, cellulose phosphate and CM-cellulose followed by gel filtrations on Bio-Gel P-100 and Sephadex G-75, and finally to a homogeneous state by SDS-polyacrylamide gel electrophoresis. The enzyme was designated RNAase 1. It was possible to detect RNAase 1 isozymes in urine and serum without difficulty using isoelectric focusing electrophoresis followed by immunoblotting with a rabbit antibody specific to RNAase 1. The existence of genetic polymorphism of RNAase 1 was detected in human serum utilizing this technique (Yasuda, T. et al. (1988) Am. J. Hum. Genet., in press). RNAase 1 in serum and urine seemed to exist in multiple forms with regard to molecular weight and pI value. Genetically polymorphic RNAase 1 was a glycoprotein, containing three mannose, one fucose, four glucosamine and no sialic acid residues per molecule, with a molecular weight of 16,000 and 17,500 determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was most active at pH 7.0 on yeast RNA substrate and inhibited remarkably by Cu2+, Hg2+ and Zn2+. It also showed definite substrate preference for poly(C) and poly(U), but much less activity against poly(A) and poly(G). Thus, the enzyme is a pyrimidine-specific RNAase.
Yasuda
T
T
Department of Legal Medicine, Fukui Medical School, Japan.
Sato
W
W
Kishi
K
K
D000596:Amino Acids / Q000032:analysis; D000918:Antibody Specificity; D002413:Cations, Divalent; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007074:Immunoglobulin G / Q000276:immunology; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000097:blood / Q000652:urine; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D009994:Osmolar Concentration; D011110:Polymorphism, Genetic; D012260:Ribonucleases / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000652:urine*; D013379:Substrate Specificity
D000596:Amino Acids; D002413:Cations, Divalent; D007074:Immunoglobulin G; D007527:Isoenzymes; D012260:Ribonucleases