S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) The Seoul Journal of Medicine The Seoul Journal of Medicine Vol. 20 No.3 (1979)
Guanine Deaminase Isoenzyme의 염색 정량 및 대사조절기전에 관한 연구
A Study on Staining, Determination and Regulatory Mechanism of Guanine Deaminasc
- 김승원; 이부영; 정홍근
- Issue Date
- 서울대학교 의과대학
- Seoul J Med, Vol.20 No.3, pp. 153-162
- 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
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.