Abstract
Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin‐like growth factor binding proteins (IGF‐BPs). IGF‐II tracer binding activity in conditioned media is two to three times greater than that of IGF‐I. Under reducing SDS‐PAGE conditions, ^125^I‐IGF affinity‐crosslinked binding protein (BP) is visualized as a broad band between 36 ± 2.9 and 49 ± 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non‐reducing conditions (33–45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity‐crosslinked binding protein exhibits a complex pattern of non‐uniform densities that suggests structural or functional IGF‐BP micro‐heterogeneity. IGF‐BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid‐sized or smaller IGF‐BPs exhibit side chains linked to the core protein with N‐glycosidic linkage. None of the crosslinked IGF‐BPs exhibit O‐linked side chains. Long‐term (12, 24, 48 hr) conditioning studies revealed that IGF‐BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short‐term (12 hr) experiments indicate that, in fresh medium, the levels of IGF‐BP increase during the first 6–8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF‐BP, aliquots of 8‐hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37°C for 16 hr. Importantly, it was found that incubation at 37°C resulted in a near total loss of binding activity. This is the first report of IGF‐BP degrading activity in a cell culture system. These findings indicate that (1) primate RPE cells rapidly secrete a complex mixture of N‐glycosylated and non‐glycosylated IGF‐BPs, and (2) the steady state levels of secreted IGF‐BP are tightly regulated at least in part through a concomitant IGF‐BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF‐BP synthesis, secretion, and degradation at the cellular level.