Protein degradation during terminal cytodifferentiation. Studies on mammary gland in organ culture.

1. In mammary gland explants subjected to experimental manipulation, average rates (during 24 h periods) of degradation of fatty acid synthase, casein and cytosol-fraction proteins were measured by a double-isotope method. Rates of degradation of fatty acid synthase were also computed from measurements of changing enzyme amount and rate of synthesis. 2. During the period of most rapid enzyme accumulation there is a transient decrease in the computed rate of degradation of fatty acid synthase. Removal of hormones produces a rapid increase in the computed rate of degradation of the enzyme. 3. The average rate of degradation of fatty acid synthase measured by the double-isotope method is low in the presence of hormones, and increases on hormone removal. This increase in degradation rate is inhibited by adrenaline and further blocked by insulin. NH4Cl (10 mM) also partially inhibits the increase in protein degradation on hormone removal. 4. The pattern of changes in the average rate of degradation of cytosol-fraction proteins is similar to that for fatty acid synthase alone. There is no relationship between subunit molecular weight and rate of degradation under all experimental conditions. 5. Isotope ratios for resolved cytosol protein mixtures are transformed logarithmically to make the standard deviations an estimate of heterogeneity of degradation rates. By this analysis, in some conditions there appears to be significant measureable heterogeneity of degradation rates. 6. Little degradation of casein is measured in the presence of hormones, but a marked increase in the rate of degradation can be measured when hormones are removed. Whereas at 24-48h NH4Cl (10 mM) has little effect on this enhanced rate of degradation, at 48-72h it causes a large decrease in degradation rate. 7. Results are discussed in terms of a two-component degradation system in mammary gland explants.

Coordination of protein synthesis and degradation.

The degree of coordination between protein synthesis and protein degradation in developing and mature cels is considered. Studies on specific enzyme and general protein turnover in developing liver and differentiating mammary gland are presented. In the mature liver mitochondrion average protein degradation rates are higher for outer membrane and intermembrane space proteins than for matrix and inner membrane proteins. Significant heterogeneity of protein degradation rates was observed only in the outer mitochondrial membrane. During postnatal development the rates of degradation of proteins in many liver cellular fractions are increased. In the mitochondrion only the average rates of degradation of proteins in the outer membrane and intermembrane space fractions increase during development. Evidence for hormonally regulated changes in both protein synthesis and degradation during mammary cell differentiation is given. The data indicate that a transitory decrease in protein degradation accompanies the increase in protein synthesis on hormonal stimulation of the tissue. The results from the two model systems are collated and used to formulate a phenomenological hypothesis of protein degradation and its integration with protein synthesis in steady-state and non-steady-state conditions.

A method for the analysis of protein turnover characteristics. Indirect estimation of rates of protein degradation.

A method for the calculation of rate constants of degradation of a specific protein during a period of change in protein amount is described. The calculation uses measurements of rates of synthesis and amount of the protein, together with one estimate of the protein half-life. The method is demonstrated by using data from measurements made on the accumulation of fatty acid synthetase in hormonally treated explants of mammary gland from mid-pregnant rabbits, and data from other authors. These calculations demonstrate a regulated programme of co-ordinated changes in rates of synthesis and degradation during a period of change in protein mass. The role of such changes in protein degradation during protein accumulation is discussed.

The role of enzyme degradation in enzyme turnover during tissue differentiation.

1. The role of enzyme degradation in enzyme turnover during tissue differentiation has been investigated by the use of equations based on the model of Berlin and Schimke (Mol. Pharmacol. 1 (1965) 149-156). 2. A digital computer has been used to calculate the change in enzyme amounts which would result from different changes in the rates of synthesis and degradation of an enzyme during cell differentiation. The energetics of these alternative processes have been compared. 3. The results demonstrate that changes in the rates of synthesis and degradation of an enzyme after a differentiation stimulus enhance the flexibility and rapidity of changes in enzyme amount during enzyme accumulation. Decreases in the rate of degradation of an enzyme during enzyme accumulation lead to a considerable saving of energy. 4. Hypotheses are proposed to account for the different modes of turnover of protein subgroups during cytodifferentiation in terms of specific changes in the rates of synthesis and degradation of the subgroups.

Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland.

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.