Diabetes mellitus is a disorder of glucose and insulin metabolism resulting in clinical manifestations including development of diabetic cataracts, retinopathy and neuropathy. Cataract formation results following increases in lens sorbitol levels as a product from conversion of glucose by aldose reductase, the first enzyme in the sorbitol pathway. There have been extensive efforts to determine ways to manipulate this enzyme, largely by the study of specific inhibitors. We have studied the physical, chemical, kinetic and structural properties of aldose reductase. Currently we are determining the primary amino acid sequence structure. We are using gas phase microsequencing techniques of peptides prepared by specific chemical and enzymatic cleavage of pure aldose reductase. The peptides are isolated and purified using HPLC and other methods. These studies are progressing at a rapid rate with the anticipated completion of most of the primary sequence structure by early in the next grant period. We then propose in these studies to determine the functional domains of the enzyme protein such as for binding sites of substrates, products, inhibitors, activators, and other effectors. We plan to look for and confirm details of predicted conformational changes on binding as these changes may play a significant role in the regulatory function of the enzyme and subsequent diabetic pathology. We will continue our efforts to establish the exact stoichiometry of basically charged centers and other residues involved in the binding of the pyridine nucleotide coenzyme. We will also use specific antibodies in identification of the functional protein domains. Many of the peptides we used in the primary sequence determinations can serve as antigens to elicit specific antibodies. We will use our peptide synthesizer to chemically prepare short peptides in critical regions of the primary sequence of as few as 6-8 amino acids or as many as 50-60 amino acids residues. Antibodies will recognize single determinates in the domain. Finally, we plan a concerted effort to obtain aldose reductase in a suitable crystalline form for X-ray diffraction studies for determination of the tertiary enzyme structure. 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.
| 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 |
