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 18 h in the presence of insulin, prolactin and cortisol were incubated at 370C for 2h in Medium 199 containing L-[4,5-3H]leucine. After a wash procedure at 40C, explants were re-incubated at 370C in fresh medium and the radioactivity of casein polypeptides isolated by isoelectric focusing (at pH4.6) 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-FU-'4C]proline and N-[ 3Hlacetyl-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 1 5 000 g, for 1 h 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 (20,ug/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 (50,uM) did not prevent the increase in radioactivity from L-[5-3HIproline 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.

Intracellular degradation of newly synthesized casein in perfused rat mammary gland

“…The fall in [3H]casein radiolabel in the absence of prolactin was reduced significantly when chloroquine was added to the perfusate after pulse labelling (Table 1). The ATP concentration in tissue freeze-clamped after the final biopsy was 0 187+ 0 022 ,umol (mg DNA)-', (n = 6), a value similar to that reported for unperfused tissue, and not significantly different to that reported for unbiopsied tissue perfused for shorter periods in the presence of insulin alone (Clegg & Calvert, 1988 (Razooki Hasan et al 1982; , and also when there is increased autocrine inhibition of milk secretion (Wilde et al 1989), a mechanism which acts to match supply of milk constituents with demand by the suckling or milking regimen (Wilde & Peaker, 1990 …”

“…Total radiolabel incorporated in the 41, 26 and 22 kDa caseins increased after [3H] proline was removed from the perfusate. Continued incorporation indicated the presence of an intracellular pool of precursor that did not exchange rapidly with unlabelled amino acid during the chase period, as was observed in mammary explants (Razooki Hasan et al 1982;. This increase in radiolabelling may also reflect the time required for [3H]proline to appear in mature (glycosylated, phosphorylated) casein.…”

“…The transient nature of this degradation, and its partial inhibition by chloroquine, indicated that it occurred through an intracellular, lysosomal mechanism. Chloroquine at higher concentrations, and also other inhibitors of lysosomal proteolysis, were shown previously to prevent casein degradation in mammary cultures (Razooki Hasan et al 1982;O'Hare et al 1986). Therefore, partial inhibition by chloroquine was probably due to use of a sub-optimal concentration, rather than involvement of another non-lysosomal mechanism.…”

“…Caseins synthesized by mammary tissue explants or cultured mammary cells are susceptible to intracellular degradation during secretion (Razooki Hasan, White & Mayer, 1982; Lee, Lee, Kaetzel, Parry & Bissell, 1985). The proportion of casein degraded depended on the stage of pregnancy or lactation at which explants were prepared, and also on the culture conditions.…”

Intracellular protein catabolism: state of the art

“…Predictably, glycosylation has a role in modulating degradation affecting lysosomal enzyme precursors and enzymes themselves in the endoplasmic reticulum and Iysosome, respectively 1551. In mammary lobulo alveolar cells secretion-coupled degradation [56] is transcriptionally and cell-shape related [59], being inversely related to secretory rate (Mayer, unpublished).…”