Interphotoreceptor retinoid-binding protein (IRBP) is rapidly cleared fromthe Xenopus interphotoreceptor matrix

The interphotoreceptor matrix (IPM) is a highly-organized extracellular mat rix critical to retinal development and function. Although the concentratio ns of its components are carefully regulated, little is known about the mec hanisms of this regulation. Interphotoreceptor retinoid-binding protein (IR BP) is the most abundant soluble protein component of the IPM. Although its rate of clearance is thought to be an important factor regulating the conc entration of IRBP within the IPM, no study has measured the rate of its ext racellular turnover. Here we determine the rate of turnover of matrix IRBP in Xenopus The rate of IRBP turnover was estimated by measuring the loss of radioactivity from protein labeled by a single injection of a radiolabeled protein precursor. To provide an estimate of the rate of IRBP turnover, we have examined the following issues: (1) Quantitative extraction of IRBP fr om the IPM for biochemical analysis. (2) Routes of delivery of radiolabeled precursor to achieve a pulse label in vivo. (3) Selection of labeled precu rsor in order to minimize reutilization of radiolabel, Using Western blot a nalysis, immunoprecipitation and immuno-electron microscopy, we found that IRBP can be quantitatively extracted from the IPM by a simple saline wash. IRBP was radiolabeled by systemic or intravitreal injection of either [S-35 ]methionine or carboxyl-terminal labeled [1-C-14]leucine, The specific acti vity of matrix IRBP was determined by either phosphorimaging or fluorograph y of Coomassie blue-stained SDS-polyacrylamide gels. Intravitreal injection of tracer was more effective than systemic delivery in achieving a pulse o f radiolabel to the retina. This may be due to intravitreal injection allow ing the body to act as a 'sink' for radiolabeled amino acid. When radiolabe led precursor was delivered by intravitreal injection, the calculated half- life of matrix IRBP using [S-36]methionine was 25.6 +/- 0.82 hr: in contras t, it was 10.7 +/- 2.9 hr using [1-C-14]leucine. The faster apparent IRBP t urnover using [1-C-14]leucine is interpreted in context of the early decarb oxylation of leucine during its degradation. Our results demonstrate rapid turnover of IRBP in the Xenopus IPM in vivo and suggest that the IPM is a d ynamic structure undergoing continuous renewal. (C) Academic Press.