583rd MEETING, CAMBRIDGE
1053
Brindley, D. N., Glenny, H. P., Pritchard, P. H., Cooling, J., Burditt, S . L. &Pawson, S. (19796) in Lipoprotein Metabolism and Endocrine Regirlotion (Hessel, L. W. & Krans, H. M. J., eds.), pp. 243-256, Elsevier/North-HollandBiomedical Press, Amsterdam Brindley, D. N., Sturton, R. G., Pritchard, P. H., Cooling, J. & Burditt, S. L. (1979~)Curr. Med. Res. Opin. 6, Suppl. 1,91-100 Clark, D. A., Leeder, L. G., Foulds, M. L. & Trout, D. L. (1970) Biochern. Pharmacol. 19, 1743-1752
De Jong, F. H. & van der Molen, H. J. (1972) J . Endocrinol. 53,461-474 Di Luzio, N. R., Stege, T. E. & Hoffman, E. 0. (1973) Exp. Mot. Pothol. 19,284-292 Dominquez, A. M., Amenta, J. S. & Palmer Saunders, J. (1963) Fed. Proc. Fed. Am. SOC.Exp. B i d . 22, 371 Glenny, H. P. & Brindley, D. N. (1978) Biochem. J . 176,777-784 Knox A. M., Sturton, R. G., Cooling, J. & Brindley, D. N. (1979) Biochem. J. 180, 441-443 Lamb, R. G. & Banks, W. L. (1977) Pliarmacologist 19, 182 Lehtonen, M. A., Savolainen, M. J . & Hassinen, I. E. (1979) FEBSLett. 99, 162-166 Pritchard, P. H., Cooling, J., Burditt, S. I,. & Brindley, D. N. (1979) J. Phnrrn. Pharmacol. 31, 406-407
Trout, D. L. (1965) Biochem. Pharmacol. 14, 813-821
Lipoprotein Lipase in Lactating Rat Mammary Tissue : Purification, Characterization and Surface Location on Isolated Mammary Cells ROGER A. CLEGG Department of Biochemistry, The Hannah Research Institute, Ayr KA6 SHL, Scotland, U.K.
The enzyme lipoprotein lipase (EC 3.1.1.34) is thought to be responsible for the harvesting of fatty acids from the triacylglycerols of circulating serum lipoproteins in those tissues that utilize these triacylglycerols (Scow et a/., 1976). The activities of lipoprotein lipaseinadiposeand mammary tissuechange markedly at parturition. Duringpregnancy, adipose-tissue activity is high and that in mammary tissue is low; after parturition, the reverse obtains (Hamosh et al., 1970). This appears to be a mechanism to avoid competition between adipose and mammary tissue in the pregnant and lactating animal for serum triacylglycerol. Other enzymes and pathways involved in lipogenesis show a similar pattern of response to that of lipoprotein lipase in these two tissues at parturition, and present evidence indicates that these adaptations are under hormonal control (Dils, 1977). Lipoprotein lipase has been isolated from rat adipose tissue (Etienne et af., 1976), but not extensively characterized. No report exists of its isolation from lactating mammary tissue of either the rat of any other mammal. The present communication outlines the preparation of lipoprotein lipase from lactating rat mammary tissue and reports on some of its properties. Purification was from an acetone/ether-dried powder of mammary tissue from 10-12day-lactating rats, and is summarized in Table 1 . The about SO-fold purified product exhibited the instability also characteristic of lipoprotein lipases from other sources: 90% of activity was lost in 24h at 4°C in 0 . 5 ~ NaCl/lOmM-sodium phosphate buffer, pH 7.2. The inclusion of glycerol (30 %, v/v) in this buffer decreased the activity loss to 47% in 24h, and lower concentrations of glycerol had proportionately smaller protective effects. The crude fractions from the early steps of the purification schedule lost activity much more rapidly than did the more purified fractions. Like its bovine milk counterpart, rat mammary-gland lipoprotein lipase aggregated at salt (NaCI) concentrations below 0.5 M. It showed, however, neither inhibition by nor cross-reaction in immunodiffusion with an antiserum against bovine milk lipoprotein lipase. The antiserum used both precipitated and inhibited bovine milk lipoprotein lipase. Rat mammary-tissue lipoprotein lipase was essentially uninhibited by 1 mM-EDTA, and proportionately inhibited up to 30% by concentrations of
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
1054
Table 1. Purification of lipoprotein lipase.from rat mammary tissue Acetone/ether-dried powdered tissue (1.8 g) was resuspended in 25 ml of 30% glycerol/ 0.5 M-NaCl/lOmM-sodium phosphate buffer, pH 7.2, and centrifuged at (30000g for 30min). The supernatant was stirred with lOml of heparin-Sepharose 4B for 30min. The heparin-Sepharose was packed into a column and eluted witha gradient of 0.5-2.0~NaCl in the above buffer.
Sample Resuspended acetone/etherdried powder 30000g supernatant Material not bound to heparin-Sepharose Active fractions from heparin-Sepharose eluate
Total protein (mg) 88 1
Total activity (nmoljmin) 23 424
Protein concn. (mglml) 23.8
590 58 1
17479 9272
20.0 12.0
12.3
24981
Specific activity (nmol/min per mg) 26.6
0.28
29.0 15.9 203 1
EDTA up to 100mM. Phenylmethanesulphonyl fluoride caused a 60% inhibition at a concentration of 2mM. The apparent molecular weight of the rat mammary-tissue enzyme (as prepared : i.e. in 30% glycerol/0.5 M-NaCl/lOmM-sodium phosphate buffer, pH7.2) was about 75000. This value was determined by exclusion chromatography on a Sephadex (3-150 column (0.5cm x 130cm) with, as calibration standards, haemocyanin (mol.wt. 3 x lo6), 8-galactosidase (mol.wt. 520000), catalase (mol.wt. 232000), lactate dehydrogenase (mol.wt. 140000), myoglobin (mol.wt. 64000) and cytochrome c (mol.wt. 12500). Sodium dodecyl sulphate/polyacrylamide-gel ( I 5 %) analysis of the purified enzyme revealed two Coomassie Blue-staining bands. The apparent molecular weights of these subunits were 16000 and 11OOO, in an approximate molar ratio (determined only from Coomassie Blue staining intensity) of 1 : 1 . Native molecular weight and subunit composition were unaffected by the inclusion of 2 m~-phenylmethanesulphonylfluoride at all stages of the preparation. The exposed surfaces of cells isolated from lactating mammary tissue by the method of Greenbaum et al. (1978) were labelled by using Na'251/lactoperoxidase/H,0,. Cell preparations were judged to be at least 90% intact by measurement of the latency of lactate dehydrogenase (EC 1.1.1.27). Of the total trichloroacetic acid-insoluble '''I incorporated, more than 98 % was membrane-bound. A buffer (IOmM-sodium phosphate, pH7.2) containing 1 % Triton X-100 and 0.5M-NaCI extracted 72 % of the membrane-bound trichloroacetic acid-insoluble '251-labelled material. Of this extract, 23% was bound to and eluted from a column (2cmx25cm) of heparin covalently linked to Sepharose 4B, in the characteristic fashion of lipoprotein lipase. Sodium dodecyl sulphate/polyacrylamide-gel analysis of this heparin-Sepharose 4B-binding material showed two bands with apparent mol.wts. of 16000 and 1 I 000,i.e. exactly the same subunit composition as was determined for the purified lipoprotein lipase from this tissue. Dils, R. R. (1977) in LipidMefnbolisrn in Mamnals, vol. 2 (Snyder, F., ed.), pp. 131-144. Plenum
Press, New York Etienne, J., Breton, M., Venhove, A. & Polonovski, J. (1976) Biochim. Biophys.
Acfa 429,
198-204
Greenbaum, A. L., Salam, A., Sochor, M. & McLean, P. (1978) Eur. J . Biochem. 87, 505-516 Hamosh, M., Clary, T. R., Chernick, S . S. & Scow, R. 0. (1970) Biochim. Biophys. A c f a 210, 473482 Scow, R. O., Blanchette-Mackie, E. J. & Smith, L. C. (1976) Circ. Res. 39, 149-162
1979
1053
Brindley, D. N., Glenny, H. P., Pritchard, P. H., Cooling, J., Burditt, S . L. &Pawson, S. (19796) in Lipoprotein Metabolism and Endocrine Regirlotion (Hessel, L. W. & Krans, H. M. J., eds.), pp. 243-256, Elsevier/North-HollandBiomedical Press, Amsterdam Brindley, D. N., Sturton, R. G., Pritchard, P. H., Cooling, J. & Burditt, S. L. (1979~)Curr. Med. Res. Opin. 6, Suppl. 1,91-100 Clark, D. A., Leeder, L. G., Foulds, M. L. & Trout, D. L. (1970) Biochern. Pharmacol. 19, 1743-1752
De Jong, F. H. & van der Molen, H. J. (1972) J . Endocrinol. 53,461-474 Di Luzio, N. R., Stege, T. E. & Hoffman, E. 0. (1973) Exp. Mot. Pothol. 19,284-292 Dominquez, A. M., Amenta, J. S. & Palmer Saunders, J. (1963) Fed. Proc. Fed. Am. SOC.Exp. B i d . 22, 371 Glenny, H. P. & Brindley, D. N. (1978) Biochem. J . 176,777-784 Knox A. M., Sturton, R. G., Cooling, J. & Brindley, D. N. (1979) Biochem. J. 180, 441-443 Lamb, R. G. & Banks, W. L. (1977) Pliarmacologist 19, 182 Lehtonen, M. A., Savolainen, M. J . & Hassinen, I. E. (1979) FEBSLett. 99, 162-166 Pritchard, P. H., Cooling, J., Burditt, S. I,. & Brindley, D. N. (1979) J. Phnrrn. Pharmacol. 31, 406-407
Trout, D. L. (1965) Biochem. Pharmacol. 14, 813-821
Lipoprotein Lipase in Lactating Rat Mammary Tissue : Purification, Characterization and Surface Location on Isolated Mammary Cells ROGER A. CLEGG Department of Biochemistry, The Hannah Research Institute, Ayr KA6 SHL, Scotland, U.K.
The enzyme lipoprotein lipase (EC 3.1.1.34) is thought to be responsible for the harvesting of fatty acids from the triacylglycerols of circulating serum lipoproteins in those tissues that utilize these triacylglycerols (Scow et a/., 1976). The activities of lipoprotein lipaseinadiposeand mammary tissuechange markedly at parturition. Duringpregnancy, adipose-tissue activity is high and that in mammary tissue is low; after parturition, the reverse obtains (Hamosh et al., 1970). This appears to be a mechanism to avoid competition between adipose and mammary tissue in the pregnant and lactating animal for serum triacylglycerol. Other enzymes and pathways involved in lipogenesis show a similar pattern of response to that of lipoprotein lipase in these two tissues at parturition, and present evidence indicates that these adaptations are under hormonal control (Dils, 1977). Lipoprotein lipase has been isolated from rat adipose tissue (Etienne et af., 1976), but not extensively characterized. No report exists of its isolation from lactating mammary tissue of either the rat of any other mammal. The present communication outlines the preparation of lipoprotein lipase from lactating rat mammary tissue and reports on some of its properties. Purification was from an acetone/ether-dried powder of mammary tissue from 10-12day-lactating rats, and is summarized in Table 1 . The about SO-fold purified product exhibited the instability also characteristic of lipoprotein lipases from other sources: 90% of activity was lost in 24h at 4°C in 0 . 5 ~ NaCl/lOmM-sodium phosphate buffer, pH 7.2. The inclusion of glycerol (30 %, v/v) in this buffer decreased the activity loss to 47% in 24h, and lower concentrations of glycerol had proportionately smaller protective effects. The crude fractions from the early steps of the purification schedule lost activity much more rapidly than did the more purified fractions. Like its bovine milk counterpart, rat mammary-gland lipoprotein lipase aggregated at salt (NaCI) concentrations below 0.5 M. It showed, however, neither inhibition by nor cross-reaction in immunodiffusion with an antiserum against bovine milk lipoprotein lipase. The antiserum used both precipitated and inhibited bovine milk lipoprotein lipase. Rat mammary-tissue lipoprotein lipase was essentially uninhibited by 1 mM-EDTA, and proportionately inhibited up to 30% by concentrations of
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
1054
Table 1. Purification of lipoprotein lipase.from rat mammary tissue Acetone/ether-dried powdered tissue (1.8 g) was resuspended in 25 ml of 30% glycerol/ 0.5 M-NaCl/lOmM-sodium phosphate buffer, pH 7.2, and centrifuged at (30000g for 30min). The supernatant was stirred with lOml of heparin-Sepharose 4B for 30min. The heparin-Sepharose was packed into a column and eluted witha gradient of 0.5-2.0~NaCl in the above buffer.
Sample Resuspended acetone/etherdried powder 30000g supernatant Material not bound to heparin-Sepharose Active fractions from heparin-Sepharose eluate
Total protein (mg) 88 1
Total activity (nmoljmin) 23 424
Protein concn. (mglml) 23.8
590 58 1
17479 9272
20.0 12.0
12.3
24981
Specific activity (nmol/min per mg) 26.6
0.28
29.0 15.9 203 1
EDTA up to 100mM. Phenylmethanesulphonyl fluoride caused a 60% inhibition at a concentration of 2mM. The apparent molecular weight of the rat mammary-tissue enzyme (as prepared : i.e. in 30% glycerol/0.5 M-NaCl/lOmM-sodium phosphate buffer, pH7.2) was about 75000. This value was determined by exclusion chromatography on a Sephadex (3-150 column (0.5cm x 130cm) with, as calibration standards, haemocyanin (mol.wt. 3 x lo6), 8-galactosidase (mol.wt. 520000), catalase (mol.wt. 232000), lactate dehydrogenase (mol.wt. 140000), myoglobin (mol.wt. 64000) and cytochrome c (mol.wt. 12500). Sodium dodecyl sulphate/polyacrylamide-gel ( I 5 %) analysis of the purified enzyme revealed two Coomassie Blue-staining bands. The apparent molecular weights of these subunits were 16000 and 11OOO, in an approximate molar ratio (determined only from Coomassie Blue staining intensity) of 1 : 1 . Native molecular weight and subunit composition were unaffected by the inclusion of 2 m~-phenylmethanesulphonylfluoride at all stages of the preparation. The exposed surfaces of cells isolated from lactating mammary tissue by the method of Greenbaum et al. (1978) were labelled by using Na'251/lactoperoxidase/H,0,. Cell preparations were judged to be at least 90% intact by measurement of the latency of lactate dehydrogenase (EC 1.1.1.27). Of the total trichloroacetic acid-insoluble '''I incorporated, more than 98 % was membrane-bound. A buffer (IOmM-sodium phosphate, pH7.2) containing 1 % Triton X-100 and 0.5M-NaCI extracted 72 % of the membrane-bound trichloroacetic acid-insoluble '251-labelled material. Of this extract, 23% was bound to and eluted from a column (2cmx25cm) of heparin covalently linked to Sepharose 4B, in the characteristic fashion of lipoprotein lipase. Sodium dodecyl sulphate/polyacrylamide-gel analysis of this heparin-Sepharose 4B-binding material showed two bands with apparent mol.wts. of 16000 and 1 I 000,i.e. exactly the same subunit composition as was determined for the purified lipoprotein lipase from this tissue. Dils, R. R. (1977) in LipidMefnbolisrn in Mamnals, vol. 2 (Snyder, F., ed.), pp. 131-144. Plenum
Press, New York Etienne, J., Breton, M., Venhove, A. & Polonovski, J. (1976) Biochim. Biophys.
Acfa 429,
198-204
Greenbaum, A. L., Salam, A., Sochor, M. & McLean, P. (1978) Eur. J . Biochem. 87, 505-516 Hamosh, M., Clary, T. R., Chernick, S . S. & Scow, R. 0. (1970) Biochim. Biophys. A c f a 210, 473482 Scow, R. O., Blanchette-Mackie, E. J. & Smith, L. C. (1976) Circ. Res. 39, 149-162
1979
