Purification and characterization of human hypoxanthine/guanine phosphoribosyltransferase.
Human hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) was purified from red blood cells by the following two methods. Method A includes (a) elimination of hemoglobin by DEAE-cellulose, (b) DEAE-Sephadex chromatography, (c) specific elution of the enzyme from CM-Sephadex by pyrophosphate and (d) Sephadex G-100 gel filtration. Method B includes (a) elimination of hemoglobin by DEAE-cellulose, (b) acid treatment at pH 4.5, (c) ammonium sulfate fractionation, (d) DEAE-Sephadex chromatography, (e) heat treatment at 85 degrees C and (f) Sephadex G-100 gel filtration. Homogeneous enzyme preparation was obtained by the two methods with 8000-9000-fold purification. The sedimentation coefficient (S20,w) was 5.5--5.6 S, and the molecular weight of the enzyme was estimated at about 85000 by the sedimentation equilibrium method. The subunit molecular weight of the untreated protein and S-carboxymethylmaleyl protein was estimated as 41000--45000 by the sedimentation equilibrium method in the presence of guanidine hydrochloride. However, the subunit size estimated by the sodium dodecylsulfate gel electrophoresis was only 26000. Amino acid composition of the enzyme was determined. Glucosamine, sialic acid and hexose were not detected in the enzyme.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004912:Erythrocytes / Q000201:enzymology*; D006454:Hemoglobins; D006461:Hemolysis; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000097:blood* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008970:Molecular Weight
D000596:Amino Acids; D006454:Hemoglobins; D046911:Macromolecular Substances; D007041:Hypoxanthine Phosphoribosyltransferase