Radioimmunoassay studies of the tissue distribution and subcellular localization of cellular retinol-binding protein in rats.

A specific radioimmunoassay for rat cellular retinolbinding protein (CRBP) was developed using antibodies raised in a turkey against purified rat testis CRBP.CRBP was radioiodinated by a modification of the Bolton-Hunter procedure.The radioimmunoassay measured CRBP levels in the range of 10 to 100 ng/assay tube.Apo-and holo-CRBP gave identical displacement curves in the radioimmunoassay.No immunological cross-reactivity was observed with either rat cellular retinoic acid-binding protein or with rat serum retinolbinding protein (RBP).In order to effectively adapt the assay to study tissue homogenates, it was necessary to add both a prot6olytic enzyme inhibitor (aprotinin) and a detergent (Triton X-100).In the absence of a detergent, most of the CRBP present in liver and testis was not immunoreactive, and was not detected in the immunoassay.However, when both aprotinin and Triton X-100 were added, homogenates of a number of tissues (liver, kidney, small intestine, testis, lung, spleen, and eye) gave '261-CRBP displacement curves which were parallel to those obtained with pure CRBP, and yielded values for CRBP content that were highly reproducible from assay to assay.The highest concentrations of immunoreactive CRBP were found in kidney and in liver.Serum, and homogenates of several tissues, including brain, muscle, fat, and skin, displayed only slight or no "%CRBP displacement in the immunoassay.Subcellular distribution studies showed that almost all (approximately 90%) of CRBP in liver was found in the soluble supernatant fraction.Most (approximately 55%) of the immunoreactive CRBP of testis was also found in the soluble supernatant fraction, although the particulate fractions of testis contained relatively more CRBP than did those of liver.The existence of an intracellular binding protein with ligand specificity for retinol was first suggested by Bashor et al. in 1973 (1).Evidence for the existence of a similar, but different intracellular protein with binding specificity for retinoic acid was reported shortly thereafter (2, 3).Since then, it has been clearly demonstrated that a number of tissues in rats, humans, and other species contain soluble proteins with binding specificity for retinol or for retinoic acid (4).