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Casein turnover in rabbit mammary explants in organ culture.

ABSTRACT: 1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.

SUBMITTER: Al-Sarraj K

PROVIDER: S-EPMC1161419 | biostudies-other | 1979 Sep

REPOSITORIES: biostudies-other

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Project description: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 degrees C for 2h in Medium 199 containing l-[4,5-(3)H]leucine. After a wash procedure at 4 degrees C, explants were re-incubated at 37 degrees 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-(3)H]proline, which is not part of casein signal peptides, was the radiolabelled precursor. 4. A dual-isotope experiment using l-[U-(14)C]proline and N-[(3)H]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 115000g(av.) 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 (20mug/ml) during the re-incubation period completely blocked the incorporation of radioactivity from l-[5-(3)H]proline into casein and the subsequent fall, indicating that observations were being made on newly synthesized casein. 8. Addition of chloroquine (50mum) did not prevent the increase in radioactivity from l-[5-(3)H]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.

Project description:1. Explants of mammary gland from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol. 2. Antibodies raised to fatty acid synthetase were used to measure the amount as well as the rate of synthesis and the rate of degradation of the enzyme in the explants over defined periods in organ culture. These measurements were also made after the hormones had been removed from the culture medium. The changes which occur in the activity of fatty acid synthetase are due to changes in the amount of the enzyme present. They are not due to activation or inactivation of the enzyme. 3. The rate of lipogenesis (measured from [1-14C]acetate) in the explants during culture varies independently of the amount of fatty acid synthetase both in the presence and after removal of the hormones. Hence the amount of fatty acid synthetase does not limit lipogenesis. The proportion of medium-chain fatty acids C8:0 and C10:0 (which are characteristic of rabbit milk) synthesized by the explants in the presence of hormones increases at about the same rate as the amount of fatty acid synthetase present. However, when hormones are removed from the medium the proportion of these acids synthesized declines as rapidly as the rate of lipogenesis and not as the amount of fatty acid synthetase presen. 4. The rates of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein in the explants were compared by measuring the incorporation of L-[U-14C]leucine into the protein of the explants. These rates increase by 5-fold and 3.6-fold respectively when explants are cultured with hormones, and they then reach approximately constant rates. When the hormones are removed there is a rapid fall in the rate of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein to values which are similar to those obtained with freshly prepared explanted tissue. 5. In unstimulated explants fatty acid synthetase appears to be degraded with a half-life of 15-21h. During the hormonally stimulated differentiation of the tissue the rate of degradation of the enzyme is considerably decreased or is switched off completely. After the amount of fatty acid synthetase has increased to a maximum the enzyme complex is again degraded with a half-life of 23-29h. The removal of hormones after the explants have been hormonally stimulated for different times results in an increase in the rate of degradation of fatty acid synthetase. However, this increase only occurs if degradation was previously proceeding at a considerably decreased rate. The degradation of the total particulate-free supernatant protein continues throughout the period of differentiation of the explant tissue in culture. It appears to be somewhat decreased during the period of rapid maturation of the tissue during culture.

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Casein turnover in rabbit mammary explants in organ culture.

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