Sato S
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Invest Ophthalmol Vis Sci. 1993 Oct;34(11):3172-8.
Recent studies have indicated that certain aldose reductase inhibitors prevent the formation of cataracts in naphthalene-fed rats. The study was designed to investigate whether aldose reductase itself is involved in the metabolism of naphthalene in the lens and the mechanism of this cataract formation.
Aldose reductase was purified from whole rat lens using a series of chromatographic steps that include gel filtration, affinity chromatography, and chromatofocusing. The dehydrogenase activity of the purified enzyme was evaluated with 1,2-dihydro-1,2-dihydroxynaphthalene (naphthalene dihydrodiol) as substrate. The same dehydrogenase activity was also examined with the recombinant protein obtained from rat lens aldose reductase clone.
Throughout the purification steps, dehydrogenase activity with naphthalene dihydrodiol as substrate coeluted with aldose reductase activity assayed with DL-glyceraldehyde as substrate. The purified aldose reductase, which appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed dehydrogenase activity with naphthalene dihydrodiol as a substrate similar to that observed with the crude extract from whole rat lens. Recombinant protein from rat lens aldose reductase clone also displayed dehydrogenase activity similar to that observed with purified rat lens aldose reductase. Both the reductase and dehydrogenase activities of purified aldose reductase were inhibited by aldose reductase inhibitors. However, inhibition of dehydrogenase activity was less than reductase activity. Aldehyde reductase, an another nicotinamide adenine dinucleotide phosphate-dependent reductase, also displayed dihydrodiol dehydrogenase activity with naphthalene dihydrodiol and this activity was also inhibited by aldose reductase inhibitors.
Aldose reductase displays dehydrogenase activity in addition to reductase activity. In rat lens aldose reductase is a major protein associated with naphthalene dihydrodiol dehydrogenase activity. This suggests that aldose reductase is linked to 1,2-dihydroxynaphthalene formation in rat lens and the subsequent formation of cataracts in naphthalene-fed rats.
最近的研究表明，某些醛糖还原酶抑制剂可预防萘喂养大鼠白内障的形成。本研究旨在调查醛糖还原酶本身是否参与晶状体中萘的代谢以及这种白内障形成的机制。
通过一系列色谱步骤（包括凝胶过滤、亲和色谱和聚焦色谱）从大鼠全晶状体中纯化醛糖还原酶。以1,2-二氢-1,2-二羟基萘（萘二氢二醇）为底物评估纯化酶的脱氢酶活性。还用从大鼠晶状体醛糖还原酶克隆获得的重组蛋白检测了相同的脱氢酶活性。
在整个纯化步骤中，以萘二氢二醇为底物的脱氢酶活性与以DL-甘油醛为底物测定的醛糖还原酶活性共洗脱。纯化的醛糖还原酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示为单一条带，以萘二氢二醇为底物显示出与大鼠全晶状体粗提物中观察到的类似的脱氢酶活性。大鼠晶状体醛糖还原酶克隆的重组蛋白也显示出与纯化的大鼠晶状体醛糖还原酶类似的脱氢酶活性。纯化醛糖还原酶的还原酶和脱氢酶活性均被醛糖还原酶抑制剂抑制。然而，脱氢酶活性的抑制小于还原酶活性。醛脱氢酶，另一种烟酰胺腺嘌呤二核苷酸磷酸依赖性还原酶，也显示出对萘二氢二醇的二氢二醇脱氢酶活性，并且该活性也被醛糖还原酶抑制剂抑制。
醛糖还原酶除了具有还原酶活性外还显示脱氢酶活性。在大鼠晶状体中，醛糖还原酶是与萘二氢二醇脱氢酶活性相关的主要蛋白质。这表明醛糖还原酶与大鼠晶状体中1,2-二羟基萘的形成以及萘喂养大鼠随后的白内障形成有关。
