Extensive destruction of newly synthesized casein in mammary explants in organ culture

Abstract

1. Explants of mammary glands of mid-pregnant rabbits that had been cultured for 18h in the presence of insulin, prolactin and cortisol were incubated at 37°C for 2h in Medium 199 containing l-[4,5-3H]leucine. After a wash procedure at 4°C, explants were re-incubated at 37°C in fresh medium and the radioactivity of casein polypeptides isolated by isoelectric focusing (at pH 4.6) was followed with time. Casein radioactivity rose during the first hour of re-incubation, but fell markedly during the subsequent hour. 2. Loss of radioactivity represented casein degradation, since less than 10% of newly synthesized casein was found in the incubation medium. 3. Such a loss of radioactivity was not due solely to hydrolysis of signal peptides, since similar results were obtained when l-[5-3H]proline, which is not part of casein signal peptides, was the radiolabelled precursor. 4. A dual-isotope experiment using l-[U-14C]proline and N-[3H]acetyl-d-mannosamine gave similar profiles of radioactivity loss from isoelectrically focused casein, indicating that degradation of mature casein was occurring. 5. Analysis of total pellet and particle-free-supernatant fractions prepared by centrifugation of explant homogenates at 115000gav. for 1h did not show loss of radioactivity on re-incubation. Total pellet-protein radioactivity remained constant, whereas total soluble-protein radioactivity increased during the 2h re-incubation period. 6. Radioactivity in a specific particle-free-supernatant polypeptide, the subunit of fatty acid synthetase, mimicked that of the total soluble protein. 7. Addition of cycloheximide (20μg/ml) during the re-incubation period completely blocked the incorporation of radioactivity from l-[5-3H]proline into casein and the subsequent fall, indicating that observations were being made on newly synthesized casein. 8. Addition of chloroquine (50μm) did not prevent the increase in radioactivity from l-[5-3H]proline into casein during the first hour of re-incubation, but did prevent the loss of radioactivity in the second hour. 9. The intracellular degradation of a newly synthesized milk protein is discussed in relation to the known intracellular degradation of other secretory polypeptides.