Nonenzymatic glycosylation of bovine retinal microvessel basement membranes in vitro. Kinetic analysis and inhibition by aspirin.

Incubation of intact bovine retinal microvessels or isolated retinal microvessel basement membranes (RVBM) with radioactive D-glucose or L-glucose, followed by basement membrane collagenous protein purification, resulted in the isolation of nonenzymatically glycosylated RVBM collagens. Type IV collagen was identified in the RVBM by selective salt fractionation, SDS-polyacrylamide gel electrophoresis, amino acid analysis, and immunoprecipitation with specific antibody. Kinetic analysis of the condensation of glucose with RVBM was carried out by labeling retinal microvessel basement membranes with D-[2-3 H]-glucose and D-[6-14 C]-glucose. The rate constant for aldimine product formation, k1, was 1.95 +/- 0.24 (SD) X 10(-4) mM-1 h-1, and the rate constant for the reversed reaction, k-1, was 5.9 +/- 1.0 X 10(-2) h-1. Based on a rate constant for the Amadori rearrangement, k2, of 8.8 +/- 1.0 X 10(-3) h-1, which was the rate-determining step, the half life of this reaction was 80 +/- 9 h. These data may be useful in estimating the glycosylation of retinal microvessel basement membranes in vivo. The nonenzymatic glycosylation of retinal microvessel basement membrane proteins was progressively inhibited by increasing concentrations (0.1 to 2.0 mM) of aspirin.