766 CYSTINOTIC FIBROBLASTS ACCUMULATE CYSTINE FROM CYSTINE BUT NOT CYSTEINE RESIDUES OF EXTRACELLULAR PROTEINS

Abstract

Cystine depleted cystinotic fibroblasts reaccumulate cystine when incubated in cystine free medium supplemented with a variety of disulfide (cystine)-containing proteins. The cystine accumulation is linear with the concentration of added protein. Correlation of the amount of degradation of 125I labelled protein with cystine accumulation yields a significant correlation (r=0.97 for bovine serum albumin [BSA], and r=0.81 for bovine insulin). Similar results have been found for human and rat albumin, and bovine y globulin & thyroglobulin. This cystine accumulation is inhibited by inhibitors of proteolysis, and inhibitors of selective endocytosis. Reduced BSA (RBSA) produced by pre-treatment with mercaptoethanol in 6 M urea yields diminished cystine accumulation even though more of the reduced protein is degraded:0.18 nmol/106 cells cystine accumulated and 0.50 mg/106 cells RBSA degraded in 24 hrs; 0.70 nmol/106 cells cystine accumulated and 0.20 mg BSA degraded in 24 hrs. Ovalbumin (OVA), which contains 4 mol of cysteine/mol of protein and 1 mol cystine yields no cystine accumulation after 24 hrs incubation in which 0.86 mg/106 cells OVA was degraded. Addition of OVA or RBSA to non-depleted cystinotic fibroblasts produces cystine depletion (30% in 4 hrs, 55% in 24 hrs). We conclude that cystinotic cells accumulate cystine through the lysosomal retention of cystine, but not cysteine residues following their release from peptide chains during proteolysis. Supported by USPHS AM 25548.