Association of matrix metalloproteinases with interphotoreceptor retinoid binding protein

Interphotoreceptor retinoid binding protein (IRBP) is one of the major components of the interphotoreceptor matrix (IPM) where it may function in retinoid transport between the photoreceptor cells and the retinal pigment epithelium. In the course of studies of the metalloproteinases (MPs) of the IPM, we found that some MPs remain associated with IRBP through the standard purification scheme. We wished to report this finding as a caution to those working with IRBP prepared from fresh tissue.

IRBP was prepared from bovine IPM by procedures commonly used for its purification, including ion exchange, concanavalin A affinity and gel filtration chromatographies. The MPs were detected by zymography, both gelatin and casein, run with and without preactivation.

Through each step of the purification both gelatinase and, especially, caseinase (stromelysin) activities were associated with IRBP. Inclusion of gelatin affinity chromatography did not totally remove the gelatinases. Much of this activity was latent and was only revealed following fractionation or by preactivation before zymography. Whether the fractionation steps remove an inhibitor or simply provide conditions appropriate for the activation of the latent zymogen forms is not known.

The close association of the MPs and IRBP may suggest a functional role for this complex. As a practical consideration, it is likely that many preparations of IRBP may be contaminated with one or more MPs. We found no evidence for any other class of proteinase. Caution should thus be exercised in using an IRBP preparation purified from fresh tissue. It should be monitored for proteinase activity by zymography and/or prepared or stored in the presence of EDTA or dithiothreitol as much as possible.