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561st MEETING, LEEDS
Effect of Hormones on the Activity of Acetyl-Coenzyme A Carboxylase in Explants of Mammary Gland from Mid-Pregnant Rabbits RAYMOND MANNING, RAYMOND DILS and R. JOHN MAYER Department of Biochemistry, University Hospitul and Medical School, Clifron Boulevard, Nottingham NG7 2UH, U.K.
Explants of mammary tissue, taken from pregnant animals and maintained in organ culture, provide a useful and convenient model system to study the hormonal initiation of lactogenesis (Forsyth, 1971 ; Forsyth et al., 1972; Strong et al., 1972; Speake et al., 1975). Lipogenesis in mammary explants from mid-pregnant (16-18 day) rabbits increases 30-4O-fold during culture for 1-4 days in the presence of insulin, prolactin and a glucocorticoid (Forsyth et al., 1972; Speake et al., 1975). This is associated with a striking increase in the synthesis of medium-chain (i.e. milk-specific) fatty acids. These changes appear to mimic the onset of milk-fat production between mid-pregnancy and subsequent lactation in the rabbit mammary gland (Smith et a/., 1968; Carey & Dils,
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Time in culture (h) Fig. 1. Effect of hormones on the specific activity of acetyl-CoA carboxylase in mammary explants during culture Mammary explants from 17-day pregnant rabbits were cultured at 37°C in an atmosphere a/., 1972) containing insulin (5,ug/ml), prolactin (1 ,ug/ml) and cortisol (1 pglml). At various times during culture, 100 explants were removed and homogenized in 0.3 ml of SOmM-Tris/HCI buffer, pH7.5, containing 1SOmM-NaC1, 10% (v/v) glycerol, 0.5 mM-EDTA and 0.5mM-dithiothreitol. The homogenate was centrifuged at 84000g,,.-min and the supernatant (which contained approx. 2-3 mg of protein/ml) was assayed for acetyl-CoA carboxylase activity (Manning et a/., 1976). Acetyl-CoA carboxylase activity of explants cultured with hormones(o), after removal of hormones ( A ) and cultured throughout in the absence of hormones (0). Values represent the means f half the difference between two independent measurements.
of C 0 2 + 0 2 (5:95) in Medium 199 (Strong et
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1972; Strong & Dils, 1972; Mellenberger & Bauman, 1974). In addition, hormonal manipulation of the tissue during culture can be used to gain valuable information about the amount and the rates of synthesis and degradation of specific proteins in the explants. Significant changes in the activity of fatty acid synthetase were observed when mammary explants from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol for periods of up to 3 days (Speake et al., 1975). By using an antiserum raised against fatty acid synthetase it was shown that variations in the enzyme activity during culture were due to changes in the amount of enzyme and not due to changes in the catalytic efficiency of a fixed amount ofenzyme. In the presence of hormones fatty acid synthetase increased to a maximum at around 40h. During the period of rapid accumulation of the enzyme the rate of synthesis was increased and the rate of degradation was considerably decreased. Removal of hormones from the culture medium resulted in a rapid decline in the rate of lipogenesis and in the proportion of medium-chain fatty acids, but only a moderate decrease ( 3 0 4 0 % ) in the amount of fatty acid synthetase. We have investigated changes in the specific activity of acetyl-CoA carboxylase during culture of explants from mid-pregnant rabbits (Fig. 1). The enzyme activity increased 16-fold in a curvilinear manner during culture for 66 h in the presence of insulin, prolactin and cortisol. Removal of hormones after 42 h resulted in a moderate decrease (approx. 50%) in the specific activity of the enzyme during the next 30h in culture. This is comparable with the decrease in specific activity of fatty acid synthetase under similar conditions (Speake et al., 1975). The enzyme activity in explants cultured for 42 h in the absence of hormones was not appreciably different from that in freshly prepared explants. During the first 42 h in the presence of hormones the specific activity of fatty acid synthetase was twice that of acetyl-CoA carboxylase, but by 66 h the specific activities of these enzymes were approximately equal. Acetyl-CoA carboxylase has been purified from the mammary gland of lactating rabbits (Manning et al., 1976) and has been used to raise an antiserum in sheep. We thank Miss J. Saxton and Mr. P. R. Golds for technical assistance. The work was supported by a grant from the Medical Research Council. Carey, E. M. & Dils, R. (1972) Biochem. J. 126, 1005-1007 Forsyth, I. A. (1971)J. Dairy Res. 3,419-445 Forsyth, I. A., Strong, C. R. & Dils, R. (1972) Biochem. J. 129,929-935 Manning, R., Dils, R. & Mayer, R. J. (1976) Biochem. J. 153,463-468 Mellenberger, R. W. & Bauman, D. E. (1974) Biochem. J. 138,373-379 Smith, S., Watts, R. & Dils, R. (1968) J. Lipid Res. 9, 52-57 Speake, B. K., Dils, R. & Mayer, R. J. (1975) Biochem. J . 148,309-320 Strong, C. R. & Dils, R. (1972) Biochem. J. 128, 1303-1309 Strong, C. R., Forsyth, I. A. &Dils, R. (1972) Biochem. J . 128,509-519
Lipid Metabolism in Cytoplasmic Droplets from Freshly Secreted Milk W. W. CHRISTIE and R. G. VERNON
The Hannah Research Institute, Ayr, KA6 SHL, Scotland, U.K. and F. B. P. WOODING
ARC Institute of Animal Physiology, Babraham, Cambridge, U.K. In 1964, McCarthy & Patton found that freshly secreted milk from goats actively incorporated isotopically labelled fatty acids into triglycerides and other lipids on incubation in uitro. The potential of this simple system for the study of triglyceride biosynthesis in uitro was investigated by Christie (1974) and it was established 1976
561st MEETING, LEEDS
Effect of Hormones on the Activity of Acetyl-Coenzyme A Carboxylase in Explants of Mammary Gland from Mid-Pregnant Rabbits RAYMOND MANNING, RAYMOND DILS and R. JOHN MAYER Department of Biochemistry, University Hospitul and Medical School, Clifron Boulevard, Nottingham NG7 2UH, U.K.
Explants of mammary tissue, taken from pregnant animals and maintained in organ culture, provide a useful and convenient model system to study the hormonal initiation of lactogenesis (Forsyth, 1971 ; Forsyth et al., 1972; Strong et al., 1972; Speake et al., 1975). Lipogenesis in mammary explants from mid-pregnant (16-18 day) rabbits increases 30-4O-fold during culture for 1-4 days in the presence of insulin, prolactin and a glucocorticoid (Forsyth et al., 1972; Speake et al., 1975). This is associated with a striking increase in the synthesis of medium-chain (i.e. milk-specific) fatty acids. These changes appear to mimic the onset of milk-fat production between mid-pregnancy and subsequent lactation in the rabbit mammary gland (Smith et a/., 1968; Carey & Dils,
0
10
20
30
40
50
60
70
80
Time in culture (h) Fig. 1. Effect of hormones on the specific activity of acetyl-CoA carboxylase in mammary explants during culture Mammary explants from 17-day pregnant rabbits were cultured at 37°C in an atmosphere a/., 1972) containing insulin (5,ug/ml), prolactin (1 ,ug/ml) and cortisol (1 pglml). At various times during culture, 100 explants were removed and homogenized in 0.3 ml of SOmM-Tris/HCI buffer, pH7.5, containing 1SOmM-NaC1, 10% (v/v) glycerol, 0.5 mM-EDTA and 0.5mM-dithiothreitol. The homogenate was centrifuged at 84000g,,.-min and the supernatant (which contained approx. 2-3 mg of protein/ml) was assayed for acetyl-CoA carboxylase activity (Manning et a/., 1976). Acetyl-CoA carboxylase activity of explants cultured with hormones(o), after removal of hormones ( A ) and cultured throughout in the absence of hormones (0). Values represent the means f half the difference between two independent measurements.
of C 0 2 + 0 2 (5:95) in Medium 199 (Strong et
VOl. 4
242
BIOCHEMICAL SOCIETY TRANSACTIONS
1972; Strong & Dils, 1972; Mellenberger & Bauman, 1974). In addition, hormonal manipulation of the tissue during culture can be used to gain valuable information about the amount and the rates of synthesis and degradation of specific proteins in the explants. Significant changes in the activity of fatty acid synthetase were observed when mammary explants from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol for periods of up to 3 days (Speake et al., 1975). By using an antiserum raised against fatty acid synthetase it was shown that variations in the enzyme activity during culture were due to changes in the amount of enzyme and not due to changes in the catalytic efficiency of a fixed amount ofenzyme. In the presence of hormones fatty acid synthetase increased to a maximum at around 40h. During the period of rapid accumulation of the enzyme the rate of synthesis was increased and the rate of degradation was considerably decreased. Removal of hormones from the culture medium resulted in a rapid decline in the rate of lipogenesis and in the proportion of medium-chain fatty acids, but only a moderate decrease ( 3 0 4 0 % ) in the amount of fatty acid synthetase. We have investigated changes in the specific activity of acetyl-CoA carboxylase during culture of explants from mid-pregnant rabbits (Fig. 1). The enzyme activity increased 16-fold in a curvilinear manner during culture for 66 h in the presence of insulin, prolactin and cortisol. Removal of hormones after 42 h resulted in a moderate decrease (approx. 50%) in the specific activity of the enzyme during the next 30h in culture. This is comparable with the decrease in specific activity of fatty acid synthetase under similar conditions (Speake et al., 1975). The enzyme activity in explants cultured for 42 h in the absence of hormones was not appreciably different from that in freshly prepared explants. During the first 42 h in the presence of hormones the specific activity of fatty acid synthetase was twice that of acetyl-CoA carboxylase, but by 66 h the specific activities of these enzymes were approximately equal. Acetyl-CoA carboxylase has been purified from the mammary gland of lactating rabbits (Manning et al., 1976) and has been used to raise an antiserum in sheep. We thank Miss J. Saxton and Mr. P. R. Golds for technical assistance. The work was supported by a grant from the Medical Research Council. Carey, E. M. & Dils, R. (1972) Biochem. J. 126, 1005-1007 Forsyth, I. A. (1971)J. Dairy Res. 3,419-445 Forsyth, I. A., Strong, C. R. & Dils, R. (1972) Biochem. J. 129,929-935 Manning, R., Dils, R. & Mayer, R. J. (1976) Biochem. J. 153,463-468 Mellenberger, R. W. & Bauman, D. E. (1974) Biochem. J. 138,373-379 Smith, S., Watts, R. & Dils, R. (1968) J. Lipid Res. 9, 52-57 Speake, B. K., Dils, R. & Mayer, R. J. (1975) Biochem. J . 148,309-320 Strong, C. R. & Dils, R. (1972) Biochem. J. 128, 1303-1309 Strong, C. R., Forsyth, I. A. &Dils, R. (1972) Biochem. J . 128,509-519
Lipid Metabolism in Cytoplasmic Droplets from Freshly Secreted Milk W. W. CHRISTIE and R. G. VERNON
The Hannah Research Institute, Ayr, KA6 SHL, Scotland, U.K. and F. B. P. WOODING
ARC Institute of Animal Physiology, Babraham, Cambridge, U.K. In 1964, McCarthy & Patton found that freshly secreted milk from goats actively incorporated isotopically labelled fatty acids into triglycerides and other lipids on incubation in uitro. The potential of this simple system for the study of triglyceride biosynthesis in uitro was investigated by Christie (1974) and it was established 1976
