초록

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담배 조직 배양으로부터 하나의 glucose-6-phosphate dehydrogenase를 정제하고 효소의 몇가지 성질을 조사하였다. 정제된 효소의 subunit 분자량을 SDS-polyacrylamide gel electrophoresis에서 60,000으로 나타났고, gel filtration에 의한 값은 117,500이었다. 따라서 이 효소는 동일한 subunit을 가진 dimer로 존재한다고 예상된다. 이 효소의 최적 pH는 8.0 근처이며 D-glucose-6-phosphate에 대한 $K_m$은 0.32 mM이었다. $NADP^+$에 대하여는 negative cooperativity 현상을 보였으며 $NADP^+$의 농도가 증가함에 따라 $K_m$과 $V_{max}$의 급격한 증가를 나타냈다. 높은 농도와 낮은 농도의 $NADP^+$에 대한 두 $K_m$ 값은 각각 0.065 mM, 0.017 mM이었다. p-chloromercuribenzoic acid, N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) 같은 sulfhydryl modifying reagents와 histidine의 imidazole기에 특이하게 반응하는 diethylpyrocarbonate가 효소의 활성을 현저히 억제한다는 사실을 보아 효소의 활성부위나 그 근처에 cysteine과 histidine이 존재할 것으로 예상된다. 한편 -SH기에 특이하게 반응하는 reagent에 대하여는 D-glucose-6-phosphate와 $NADP^+$가 모두 보호작용을 나타내었고 imidazole기에 특이하게 반응하는 reagent에 대하여는 $NADP^+$만이 보호작용을 나타내었으므로 cysteine과 histidine이 substrate와 coenzyme binding에 필수적인 아미노산이라고 사료된다.

Abstract ▼ AI-Helper

A method for the purification of D-glucose-6-phosphate dehydrogenase from tobacco callus was developed. The purified enzyme has the subunit molecular weight of 60,000 as determined by SDS polyacrylamide gel electrophoresis. The molecular weight of the enzyme as estimated by gel filtration on Sephade...

A method for the purification of D-glucose-6-phosphate dehydrogenase from tobacco callus was developed. The purified enzyme has the subunit molecular weight of 60,000 as determined by SDS polyacrylamide gel electrophoresis. The molecular weight of the enzyme as estimated by gel filtration on Sephadex G-150 was about 117,500. The $K_m$ value for glucose 6-phosphate was 0.32 mM. The $NADP^+$ double reciprocal plot contained an abrupt transition between two linear sections with an abrupt increase in $K_m$ and $V_{max}$ values as the concentration of $NADP^+$ increase. The two $K_m's$ for high and low $NADP^+$concentrations were 0.065 mM and 0.017 mM, respectively. The sulfhydryl modifying reagents such as p-chloromercuribenzoic acid, N-ethylmaleimide and 5,5'-dithiobis (2-nitrobenzoic acid) inhibited the enzyme activity, and the inhibition by these reagents was protected by either glucose-6-phosphate or $NADP^+$. Imidazole group directed reagent such as diethylpyrocarbonate also inhibited the enzyme activity and the inhibition was protected by $NADP^+$ alone. These facts suggest that cysteinyl residue and histidyl residue are essential amino acids for function in this enzyme.