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.
Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.