NONENZYMATIC GLYCATION--ENZYMATIC MECHANISM FOR CONTROL

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DESCRIPTION: (Adapted from the applicant's abstract) Diabetic complications remain a serious and intractable problem in the management of diabetes mellitus. The nonenzymatic glycation hypothesis postulates that diabetic complications are a result, at least in part, of nonenzymatic modifications of proteins by glucose. The initial product in this process is fructoselysine (FL) which subsequently undergoes a series of further rearrangements and reactions to form the Advanced Glycation Endproducts (AGEs). This proposal intends to test the hypothesis that FLPT is part of an intracellular deglycating system. The research plan consists of three Specific Aims: 1) Using intact cell incubations, test of the role of FLPT by comparing the amount of nonenzymatic glycation produced by glucose vs. that produced by sugars which are not substrates of FLPT; 2) Purify FLPT from human erythrocytes and characterize it fully in terms of its functional and structural properties, and 3) Initiate work to purify an apparent companion enzyme of FLPT, FL3P-hydrolase. The experiments described under the first aim will use erythrocytes (diluted to 0.5% hematocrit), hemoglobin that has been "deglycated" by passage through phenylboronate, and vascular endothelial cells. The latter are included because the investigator believes that lysine-rich histones are an important glycation target and that, by assessing growth, protein and DNA synthesis, he may show that sugars not metabolized by FLPT will have a greater inhibitory effect than glucose on these parameters. He refers to the first two systems as in vivo and in vitro, but these are misnomers. Incubation of red cells, especially heavily diluted, does not equate with the in vivo situation. Hemoglobin passed through PBA will be conformationally distorted and denatured. Conclusions regarding the existence of a FLPT system based on the amount of glycated product generated after incubation of erythrocytes vs. processed hemoglobin with different sugars would be fraught with error. It is parenthetically noted that the same confusion is shown in the background section wherein the investigator cites differences in sites of hemoglobin glycation in vitro vs. in vivo as supporting the existence of a FLPT-deglycation system, without regard to the fact that the in vitro situation imposes changes in accessibility, local pKa, etc. that govern the susceptibility of lysine residues to glycation. The enzyme purification schedules are straightforward and a sample of purified FLPT has been submitted for amino acid analysis. The investigator has set up functioning assays for FLPT and FL3P activity that are used to monitor the purification process.