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KMID : 0378019800230050071
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New Medical Journal
1980 Volume.23 No. 5 p.71 ~ p.82
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Properties of Guanine Aminohydrolase Isoenzymes from Rat Brain
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Abstract
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Guanine aminohydrolase (Guanine deaminase(GDA), EC 3.5.4.31 was partially purified approximately 10 fold from the supernatant of rat brain homogenate by salting-out, followed by dialysis and DEAF-cellulose column chromatography.
The GDA activities were resolved into two fractions (A and B peak activities) on DEAE-cellulose column. Fraction A was the first and sharp peak, whereas Fraction B, the second and broad one.
To characterize the isoenzymic nature of each fraction, the kinetic properties were observed and the GDA isoenzymes were visualized on the polyacrylamide gel after electerophoresis.
The results obtained are summarized as follows:
1. GDA isoenzymes were stained on the polyacrylamide gel by the coupled reaction of GDA and xanthine oxidase, a flavoprotein, in the presence of phenazine methosulf ate, an electron carrier and nitroblue tetrazolium as an electron acceptor.
2. GDA of Fraction A was further separated into two bands on the elect ropherogram. One of them was a broad, intensely colored band with the slowest electrophoretic mobility which was not shown in the electropherogram of the supernatant of rat brain homogenate.
3. GDA of Fraction B was again further separated into 6 broad bands on the electropherogram.
4. GDA of Fraction A showed a signoidal response to the substrate, guanine, and a hyperbolic response to 8-azaguanine. Its apparent Km values were 12pM as a (SD 0. 5 value for guanine and 105uM for 8-azaguanine.
5. GDA of Fraction B revealed a hyperbolic response to guanine and 8-azaguanine. Its apparent Km values were 7.6pM for guanine and 100pM for 8-azaguanine respectively.
6. The GDA activities of both fractions were unaffected with 0. 1mM or O.01mM concentration of xanthine, ammonia, allantoin, GMP, GDP and GTP and also with Mg++ Ca++, Lit, Cu++. and Fe+++ inhibited the GDA activities of both fraction.
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KEYWORD
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