PubMed · 9434468
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N Hotta. 1997. [Epalrestat].. https://pubmed.ncbi.nlm.nih.gov/9434468/
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A xylose reductase (XR) gene was identified from the Neurospora crassa whole-genome sequence, expressed heterologously in Escherichia coli, and purified as a His6-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.
The aim of the present study was to evaluate the effect of iron overload on gene expression in HepG2 cells by differential display. Iron-treated cells showed a 50% decrease in apolipoprotein B100 (Apo B100) and a 2- and 3-fold increase in semaphorin cd100 and aldose reductase mRNA, respectively, with parallel variations in Apo B100 and aldose reductase proteins. These effects were time-dependent. Vitamin E prevented the increase in aldose reductase expression, but had no effect on Apo B100 and semaphorin cd100. Treatment with hydrogen peroxide and 4-hydroxy-2,3-nonenal increased only aldose reductase mRNA. These data suggest that iron can affect mRNA levels by lipid peroxidation-dependent and -independent pathways.
A method for the isolation of an intact, non-covalent complex formed by the interaction of aldose reductase, NADP(H) nucleotide, and inhibitor has been developed to aid in the discovery and development of novel aldose reductase inhibitors. In the complexes isolated, both the carboxylic acid-containing inhibitor tolrestat and the spirohydantoin-containing inhibitor AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione) tightly bound in a 1:1 ratio to aldose reductase complexed with either NADPH or NADP+. Inhibitor binding to either the enzyme-NADP+ or enzyme-NADPH complex appeared to be equal and pH-dependent, with maximum binding observed at a pH range of 7 to 8.5 where the inhibitors are ionized. These results indicated that the charge state of the cofactor (NADPH vs NADP+) is not critical for inhibitor binding to aldose reductase. Molecular modeling studies suggested that His110 plays a crucial role in directing charged inhibitors containing either a carboxylate or an ionizable hydantoin group to the active site of aldose reductase by providing charge interaction.