Proteins iodinated by the Chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis

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Abstract
Proteins labeled with either 3H by reductive methylation or 125I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin [BSA] and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. 125I-labeled proteins were apparently degraded at rates far exceeding their 3H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3 h incubation, 3-5 times more 125I-labeled BSA and X. laevis serum proteins lacking albumin were degraded than the corresponding 3H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24 h incubation, the degradation of 125I-labeled vitellogenin was 15 times greater than that of [3H]vitellogenin labeled in vivo (60% compared to 4%). Examination of the relative amounts of 3H- compared to 125I-labeled BSA deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable 3H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the 3H-labeled ligands. Passage of dissolved oocytes previously exposed to 125I-labeled BSA (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of BSA to intermediate MW components and the presence of a large amount (.apprxeq. 90%) of labeled low MW compounds, which analysis showed to be 72% free I. The evolution of either iodotyrosine or free I would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). Apparent degradation rates observed for endocytotically incorporated proteins may vary, depending on the method used to label the protein, and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.