쥐의 간 Cytosolic Aldehyde Reductase의 정제 및 특성에 관한 연구 = Purification and Characterization of Rat Liver Cytosol Aldehyde Reductase

국문 초록 (Abstract)

쥐의 간 시토졸 분획에서 두 가지 aldehyde reductase [ALR: EC 1.1.1.2J를 분리하였는데, 그 중 p-nitrobenzaldehyde에 대한 $K_m$값이 높은것(high $K_m$, ALR)은 황산암모늄 분별침전, DEAE-Sephacel, hydroxyapatite, blue sepharose CL-6B chromatography 방법을 이용하여 분리 정제하였고, $K_m$값이 낮은것(low $K_m$ ALR)은 활성이 적고 불안정하여 부분 정제하여 특성을 조사하였다. High $K_m$ ALR은 SDS-PAGE상에서 42,000 dalton의 단일띠로 나타났으며 superose 12 column을 이용한 FPLC gel filtration을 수행한 결과 분자량이 44,000 dalton의 monomer임이 확인되었다. High $K_m$, ALR의 최적 pH는 6.5, pI는 6.2이었고 low $K_m$ ALR의 최적 pH는 5.5이었다. 두 ALR 모두 $37^{\circ}C$ 에서 안정하였으나 $54^{\circ}C$ 이상에서는 활성이 급격히 저하되었다. High $K_m$, ALR. low $K_m$ ALR은 모두 p-nitrobenzaldehyde에 대해 좋은 반응성을 가쳤고 특히 high $K_m$ ALR은 succinic semialdehyde에 대해 높은 반응성을 나타낸 것이 특색이다. 그러나 high $K_m$ ALR과 low $K_m$, ALR의 기질 특이성에는 상당한 차이가 있었다. High $K_m$, ALR은 보조효소로 NADPH만을 이용하지만 low $K_m$, ALR은 NADH, NADPH를 모두 보조효소로 이용한다. 두 효소는 $Mg^{2+}$, $Li^+$, $Zn^{2+}$, $Ca^{2+}$에 의한 영향을 받지 않았으나, high $K_m$, ALR은 $Cu^{2+}$에 의해 활성이 억제되었는데, low $K_m$, ALR은 반대로 $Cu^{2+}$에 의해 활성이 촉진되었다. Barbital은 high $K_m$, ALR에 대해 비경쟁적 억제작용을 나타내고 $K_i$값은 $35{\mu}M$이였으며, indole-3-acetate에 의해서는 반경쟁적인 억제현상을 나타내고 $K_i$값은 $496{\mu}M$이었다.

다국어 초록 (Multilingual Abstract)

Aldehyde reductase (ALR) isozymes of rat liver cytosol fraction were separated and the ALR having high $K_m$ $(101{\mu}M)$ for p-nitrobenzaldehyde was purified by the methods of ammonium sulfate precipitation, DEAE-Sephacel chromatography, hydroxyapatite chromatography and Blue-Sepharose CL-6B affinity chromatography. The ALR having low $K_m$ $(29{\mu}M)$ for p-nitrobenzaldehyde was only partially purified because of its instability and low activity. The molecular weight of high $K_m$ ALR was determined to be 44,000 dalton by Superose gel filtration in FPLC system. SDS-polyacrylamide gel electrophoresis showed that it was monomer having molecular weight of 42,000 dalton. The optimal pH of high $K_m$ ALR was 6.5 and that of low $K_m$ ALR was 5.5. The isoelecric point of high $K_m$ ALR was 6.2. The above two isozymes were very stable at $37^{\circ}C$, but their activities decreased rapidly as the temperature increased to $54^{\circ}C$. The good substrate of the above two isozymes was found to be p-nitrobenzaldehyde, but their substrate specificity was very different from each other. For low $K_m$ ALR, both NADH and NADPH were used as an coenzyme but high $K_m$ ALR used only NADPH as a coenzyme. It was also realized that $Cu^{2+}$ inhibited high $K_m$ ALR activity but stimulated low $K_m$ ALR activity. Barbital was shown to act as noncompetitive inhibitor of high $K_m$ ALR and its $K_i$ was $35{\mu}M$. Indole-3-acetate was shown to act as uncompetitive inhibitor of high $K_m$ ALR and its $K_i$ was $496{\mu}M$.