Diabetes mellitus is a disorder of glucose and insulin metabolism resulting in a variety of clinical manifestations including development of diabetic cataracts, retinopathy and neuropathy. Cataract formation results following increases in lens sorbitol levels which are formed as a product from conversion of glucose by aldose enzyme reductase, the first in the sorbitol pathway. Hence, there have been extensive efforts to determine ways to manipulate this enzyme, largely by the study of specific inhibitors. Our role in these efforts has been in recent years, to determine the physical, chemical, kinetic and structural properties of aldose reductase. We have begun the determination of the primary amino acid sequence using bovine lens aldose reductase. The primary structure determination of the enzyme will be continued by conventional sequencing of the cyanogen bromide and trypsin peptides obtained from aldose reductase. In addition the enzyme will be obtained by cloning the recombinant DNA in a microbial vector. DNA recombinant techniques will also be used in the primary structure determination. The anion recognition sub-site and the aromatic sub-site for binding NADPH will be determined after quantitation of the number of participating residues. These sites will be covalently labeled by reporter groups so that further identification of the active site involved in coenzyme binding can be determined. The sugar substrate binding site will be investigated in the same way. Physical studies involving circular dichroism and Raman spectra will be undertaken, as will X-ray diffraction studies to determine the secondary and tertiary structure at the enzyme active site. These studies will be helpful in detection of structural conformational changes that may be correlated with changes in enzyme activity induced by natural activators as NADPH or inhibitors and other effectors. We believe these approaches to the study of the structure and regulation of this enzyme will lead to a more precise understanding and control of the pathological effects observed in diabetes. This knowledge will be useful to other investigators studying the secondary complications of diabetes, especially those who are designing drugs, to allow them to choose suitable modifications of existing drugs or to select other drugs which might be more specific for aldose reductase and have decreased undesirable side reactions.
| Schade, S Z; Early, S L; Williams, T R et al. (1990) Sequence analysis of bovine lens aldose reductase. J Biol Chem 265:3628-35 |
| Lee, S M; Schade, S Z; Doughty, C C (1985) Aldose reductase, NADPH and NADP+ in normal, galactose-fed and diabetic rat lens. Biochim Biophys Acta 841:247-53 |
