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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.

biochemical journal. cellular aspects

Regulation of enzyme turnover during tissue differention. Studies on the effects of hormones on the turnover of fatty acid synthetase in rabbit mammary gland in organ culture