Guanine Deaminase Isoenzyme의 염색 정량 및 대사조절기전에 관한 연구

Abstract

For preparation of the isoenzymic forms of guanine deaminase(GDA). the supernatant from rat liver homogenate was subjected to salting-out, followed by dialysis and DEAE-cellulose column chromatography. The fractions collected from the column were pooled into Franction A, Band C according to the chromatographic elution pattern. To characterize the isoenzymic nature of each fraction, the enzyme kinetic properties were studied and the isoenzymcs were visualized on the polyacrylamide gel after electrophoresis. The results obtained are summarized as follows: I. GDA isoenzymes were stained by coupling with a flavoproetein, xanthine oxidase in the presence of an elecron carrier, phenazinc methosul£atc and nitro blue tetrazolium which in turn is reduced to a water-insoluble dye, formazan. 2. Fraction A was the first-eluted peak of GDA activities from the DEAE-cellulose column and was separated into two major bands on the polyacrylamide gel after electrophoresis which showed slow migration through the gel. The slowest-moving band of Fraction A was not shown in the electropherogram of the 105,000 x g supernatant. Fraction A showed a sigmoidal response to the substrate concentration of guanine and its apparent Km value was 18pM. 3. Fraction B was the second-eluted peak from the column and was stained as a diffuse hand on the clcctropherogram which showed intermediate migration through the gel. Fraction B revealed a hyperbolic response to the substrate concentration of guanine and its apparent Km value was 22pM. 4. Fraction C was the lastly eluted fractions from the column and was separated into two diffuse bands on the gel which showed the most rapid migration through the gel. Fraction C revealed a hyperbolic response to the substrate concentration of guanine and its apparent Km value was 12pM. 5. All fractions were unaffected at O.lmM concentration of xanthine, hypoxanthine, NH., allantoin, GMP, GDP and GTP.