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Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart
ANALYTICAL
BIOCHEMISTRY
135, 197-200 (1983)
Rapid Preparation Lipoprotein
of a Relatively Stable, Partially Purified Lipase from Perfused Rat Heart
LOUISE BRISSETTEAND
SIMON-PIERRE
NOEL
Dipartement de Biochimie. Universitk de Montrt!al, MontGal H3C 337 Canada Received April 25, 1983 Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M Nail containing 16 U of heparin and 10?J0glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or posthepatin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37’C and retained 75% of its activity after 10 days at 4°C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.
Lipoprotein lipase (LPL)’ (EC 3.1.1.34) plays an important role in the catabolism of circulating triacylglycerol (TG)-rich lipoproteins (1). In order to investigate its action, TGrich lipoproteins have been subjected to in vitro catalysis with LPL from various sources such as postheparin plasma (PHP) (2-4), acetone powder of rat heart (5) or adipose tissue (6,7), perfused rat heart (8-l 1) or adipose tissue (12), and cow’s milk lipase ( 13,14). Depending on the purpose, these preparations may have unwanted features. For instance, PHP not only contains LPL but also all the other plasma constituents. Perfusion of animal tissues may lead to substantial uptake of the lipolysis products ( 15,16) while acetone powder of rat hearts has low lipase specific activity suggesting the presence of many contaminating proteins. Several investigators have taken advantage of the known property of heparin to release endothelium-bound LPL in order to obtain ’ Abbreviations used: LPL, lipoprotein lipase; TG, triacylglycerol; PHP, postheparin plasma.
the enzyme in its soluble form ( 16- 18). However, the enzyme preparation thus obtained was often used as a convenient source of LPL without further characterization. In this study we use the same heparin solution to perfuse several rat hearts in order to quickly obtain a preparation enriched in LPL while in a relatively small volume (5 ml). When compared with other rapid methods of preparing soluble rat LPL, our enzyme preparation has a much higher specific activity indicating that lipase enrichment was achieved without the need of undesirable long steps of purification. Moreover, we show that the enzymatic activity remains stable for at least an hour at 37°C which makes this LPL preparation suitable for metabolic studies in vitro. MATERIALS
AND METHODS
Experimental animals. All rats were male Sprague-Dawley retired breeders weighing 500-600 g, bought from Canadian Breeding Farm and Laboratories, St. Constant, Qu&ec. They were maintained in the 12-h light-dark197
0003-2697183 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reprcdutimn m any form reserved.
198
BRISSETTE
ness cycle and fed ad libiturn normal rat chow (Ralston Purina Co., St. Louis, MO.). Animals were anesthetized with diethyl ether for heart removal and with sodium pentobarbital (60 mg/kg body wt) for PHP collection. Lipoprotein lipase preparations. PHP was prepared as previously described (4). Acetone powder of heart tissue was prepared from overnight fasted rat hearts according to the method of Kom (5). Heparin-perfused rat heart LPL was obtained as follows: hearts were excised from fasted rats and the aorta was connected to a 161/z-gauge needle which was attached to the bottom opening of a three-way stopcock coupled to a 50-cm column filled with 0.15 M NaCl (saline) at room temperature. As soon as the heart was attached, the flow of saline from the column was immediately started. After 15 to 30 s of initial washing, 50 ml of ice-cold saline was perfused through the heart with a syringe fitted to the middle opening of the stopcock in order to carefully wash out most of the residual blood from the cardiac vascular space. Special care was taken to fill up the middle opening with saline from the column to remove all air bubbles before connecting the 50-ml syringe. After this extensive washing treatment, the hearts were slowly perfused using a syringe with 5 ml ice-cold saline containing 16 U of heparin and 10% glycerol (average flow rate of 1 ml/min). The LPLcontaining outflow was collected in a beaker kept on ice and reused to perfuse several washed hearts (up to 6) in order to enrich the preparation in lipase activity. Enzymatic assay. The enzyme activity of the LPL preparations was measured by using [9, 10-3H(N)]triolein (New England Nuclear, Boston, Mass., sp act 100-l 50 Ci/mmol) emulsion in glycerol as described by NilssonEhle and Schotz (19). The liberated free fatty acids were extracted (20) and counted in toluene-based scintillating solution with 40% efficiency. The enzymatic lipase activity of 1 mU was defined as the production of 1 nmol of oleic acid per minute at 37°C. Analytical determinations. The proteins
AND NOifL
were measured by the method of Lowry et al. (2 l), using bovine serum albumin as standard. Total lipids were extracted (22), separated by thin-layer chromatography, and determined as described (4). When needed, the LPL-containing solution was concentrated by using Aquacide II-A (Calbiochem-Behring Corp., La Jolla, Calif.). RESULTS
We have used the well-known property of heparin to release endothelium-bound LPL from perfused rat hearts (18). In order to get a LPL preparation with high specific activity, rat hearts were first extensively washed with 0.15 M NaCl to get rid of most of the serum proteins. Following this treatment, up to 6 hearts were perfused with the same heparin solution to enrich the preparation with LPL activity. We noted that the heparin solution gradually lost its ability to release cardiac LPL when more than 6 hearts were perfused with the same solution. If LPL from more than 6 hearts need to be prepared, more heparin should be added to the perfusate. This procedure was compared with two other rapid methods for preparing rat heart LPL. Table 1 shows that LPL from hearts perfused with a heparin solution containing 10% glycerol had a much higher specific activity than the rat heart acetone powder and the rat PHP. When glycerol was omitted from the heparin perfusate, the total LPL activity released from the heart was decreased 2.5-fold: 57.4 f 12.2 mU/g of heart with 10% glycerol versus 22.7 + 3.7 mU/g of heart without glycerol (mean + SEM, n = 3). The enzyme activity was also more stable when stored at 4°C if 10% glycerol was added to the perfusion medium. Figure 1 shows that in the absence of glycerol, the cardiac LPL lost 50% of its original activity after 3 days. In contrast, in the presence of 10% glycerol, only 25% of the LPL activity was lost during the first 2 days and it then remained stable for at least 8 more days. At 37”C, the glycerol preparation was also stable for 1 h (Table 2).
RAT TABLE
HEART
LIPOPROTEIN
TABLE
1
SPECIFICACTIVITY OF RAT LIPOPROTEIN LIPASE OBTAINED BY THREE DIFFERENT METHODS
Method
Specific activity” (mU/ mg protein)
Relative specific activity
99 *
34 (4)
1
378 f
28 (3)
4
4251 + 318 (3)
43
’ The results are the means f SEM of the number of experiments indicated in parentheses. The mU is defined as 1 nmol of free fatty acid liberated per minute at 37°C. ’ Fifty milligrams of acetone powder was dissolved in 1 ml of 25 mM ammonium hydroxide according to Kom (5). ‘The rat hearts were thoroughly washed with 50 ml of cold 0.15 M NaCl before perfusion with the heparin solution and perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol.
The activity, however, declined rapidly to less than 50% in the next 30 min. Even though the hearts were carefully perfused with 50 ml of saline, trace amounts of lipids could still be consistently detected in the LPL-containing perfusate, especially when several hearts were perfused sequentially with the same heparin solution. In order to evaluate the extent of lipid contaminants, the eluate
2
STABILITY OF THE PARTIALLY PURIFIED HEART LPL AT 37°C’ Time of incubation at 37°C (min) 0
Rat heart acetone powder b Rat postheparin plasma Heparin-perfused’ rat heart
199
LIPASE
Relative LPL activityb (% of zero time)
10
100.0 115.7 f
20 30 40 50 60
98.3 108.3 91.7 85.7 88.0
+ f + + +
3.2 (4)
13.9 (3) 13.5 (4) 7.5 (3) 14.1 (3) 14.0 (4)
a Washed hearts were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol. The LPL activity was measured by using a tritiated triolein emulsion as described under Materials and Methods. b The results are the means f SEM of the number of experiments indicated in parentheses.
from 10 perfused hearts was extracted and the lipids were separated by thin-layer chromatography. The mass of triacylglycerol, free and esterified cholesterol, as well as phospholipid, was determined. We calculated that each heart contributed approximately 6 to 9 nmol of each lipid class. Using electron microscopy at a magnification of 42,000 we failed to detect any lipoprotein particles in an enzyme preparation (from 10 hearts) concentrated 50 times. DISCUSSION
“0 8 *5’ t
I I I
FIG. 1. Stability of the perfused rat heart LPL at 4°C with (----) or without (- - -) 10% glycerol in the perfusion medium. Each point represents the mean of three determinations. Emymatic activity measured immediately after perfusion was taken as 100%.
The activity of purified rat heart LPL is particularly unstable at all temperatures. Ethylene glycol(1 M) appears to delay the decrease in enzyme activity at 4°C while glycerol (20%) somewhat stabilizes it at -20°C; the purified enzyme retained 56% of its initial activity after 7 days at -20°C (23). In this paper we present a partial characterization of a soluble rat heart LPL preparation which has both a relatively high specific activity and a reasonable stability. The observed stability at 37°C for 1 h is in agreement with the findings of Greten et al. (7), who have shown that PHP kept its lipase activity during 60 min incubation at 37°C. Like others, we observed that glycerol has
200
BRISSETTE
a stabilizing effect on the LPL activity at 4°C (Fig. 1). Furthermore, the presence of glycerol in the heparin perfusate increased 2.5fold the total enzyme activity releasable from the hearts. The concomitant release of nonspecific proteins was only increased 1.5-fold; therefore the specific activity of LPL was increased in the presence of glycerol. Glycerol appears to have two additive effxts: (a) it may promote the release of additional proteins (including LPL) from perfused rat hearts; (b) it may also stabilize the enzyme since the increment of nonspecific proteins was lower than that of total apparent LPL activity. Similarly, Crass and Meng (24) observed that the addition of 10% rat serum to the perfusate increased the release of total LPL activity from rat hearts. They ruled out the activation effect of apolipoprotein C-II since addition of 10% serum to the LPL solution (prepared in absence of serum) did not account for the observed increase. These authors concluded that serum appeared to favor the release of LPL from the heart tissue and not to activate or stabilize the enzyme. ACKNOWLEDGMENTS We thank Mireille Fyfe for assistance during heart perfusions and Marcel Smit for drawing the figure. The help of Rose-Mai Roy in typing the manuscript is greatly ap preciated. This work was supported by the Quebec Heart Foundation. L. Brissette is a recipient of a studentship from the Canadian Medical Research Council.
REFERENCES 1. Robinson, D. S. ( 1970) in Comprehensive Biochemistry (Florkin, M., and Stotz, E. W., eds.), Vol. 28, pp. 51-l 16, Elsevier, Amsterdam.
AND NOEL 2. Bilheimer, D. W., Eisenh S., and Levy, R. E. (1972) Biochim. Biophys. Acta 260, 212-221. 3. Eisenberg, S., and Rachmilewitz, D. (1975) J. Lipid Res. 16, 341-351. 4. Ndl, S-P., and Rubinstein, D. (1981) Canad. J. Biochem. 59,447-453. 5. Kom, E. D. (1955)J. Biol. Chem. 215, l-14. 6. Kom, E. D., and Quigley, T. W., Jr. (1957) J. Biol. Chem. 226, 833-838. 7. Greten, H., Levy, R. I., and Fredrickson, D. S. ( 1968) B&him. Biophys. Acta 164, 185-194. 8. Noel, S.-P., Dolphin, P. J., and Rubinstein, D. ( 1975) B&hem. Biophys. Res. Commun. 63, 764-77 1. 9. Chajek, T., and Eisenberg, S. (1978) J. Clin. Invest. 78, 1654-1665. 10. Pedersen, M. E., Wolf, L. E., and Schotz, M. C. ( 198 1) Biochim. Biophys. Acta 666, 191-199. 11. Fielding, C. J., and Higgins, J. M. (1974) Biochemistry 13,4324-4330. 12. Fielding, C. J. (1976) Biochemistry 15, 879-884. 13. Eisenbetg, S., Feldman, D., and Olivecrona, T. (198 1) B&him. Biophys. Acta 665,454-462. 14. Deckelbaum, R. J., Olivecrona, T., and Fainaru, M. (1980) J. Lipid Res. 21, 425-434. 15. Dory, L., Pocock, D., and Rubinstein, D. (1978) Biochim. Biophys. Acta 528, 161-175. 16. Tam, S. P., Dory, L., and Rubinstein, D. (1981) J. Lipid Res. 22, 64 l-65 1. 17. Ben-Zeev, O., Schwalb, H., and Schotz, M. C. (1981) J. Biol. Chem. 256, 10,550-10,554. 18. Fielding, C. J., and Higgins, J. M. (1974) Biochemistry 13,4324-4330. 19. Nilsson-Ehle, P., and Schotz, M. C. (1976) J. Lipid Res. 17, 536-541. 20. Trout, D. L., Estes, E. H., and Friedberg, S. J. ( 1960) J. Lipid Res. 1, 199-202. 21. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275. 22. Folch, L., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509. 23. Twu, J.-S., Garhnkel, A. S., and Schotz, M. C. (1975) Atherosclerosis 22, 463-472. 24. Crass, M. F. III, and Meng, H. C. (1964) Amer. J. Physiol. 206, 6 10-6 14.
BIOCHEMISTRY
135, 197-200 (1983)
Rapid Preparation Lipoprotein
of a Relatively Stable, Partially Purified Lipase from Perfused Rat Heart
LOUISE BRISSETTEAND
SIMON-PIERRE
NOEL
Dipartement de Biochimie. Universitk de Montrt!al, MontGal H3C 337 Canada Received April 25, 1983 Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M Nail containing 16 U of heparin and 10?J0glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or posthepatin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37’C and retained 75% of its activity after 10 days at 4°C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.
Lipoprotein lipase (LPL)’ (EC 3.1.1.34) plays an important role in the catabolism of circulating triacylglycerol (TG)-rich lipoproteins (1). In order to investigate its action, TGrich lipoproteins have been subjected to in vitro catalysis with LPL from various sources such as postheparin plasma (PHP) (2-4), acetone powder of rat heart (5) or adipose tissue (6,7), perfused rat heart (8-l 1) or adipose tissue (12), and cow’s milk lipase ( 13,14). Depending on the purpose, these preparations may have unwanted features. For instance, PHP not only contains LPL but also all the other plasma constituents. Perfusion of animal tissues may lead to substantial uptake of the lipolysis products ( 15,16) while acetone powder of rat hearts has low lipase specific activity suggesting the presence of many contaminating proteins. Several investigators have taken advantage of the known property of heparin to release endothelium-bound LPL in order to obtain ’ Abbreviations used: LPL, lipoprotein lipase; TG, triacylglycerol; PHP, postheparin plasma.
the enzyme in its soluble form ( 16- 18). However, the enzyme preparation thus obtained was often used as a convenient source of LPL without further characterization. In this study we use the same heparin solution to perfuse several rat hearts in order to quickly obtain a preparation enriched in LPL while in a relatively small volume (5 ml). When compared with other rapid methods of preparing soluble rat LPL, our enzyme preparation has a much higher specific activity indicating that lipase enrichment was achieved without the need of undesirable long steps of purification. Moreover, we show that the enzymatic activity remains stable for at least an hour at 37°C which makes this LPL preparation suitable for metabolic studies in vitro. MATERIALS
AND METHODS
Experimental animals. All rats were male Sprague-Dawley retired breeders weighing 500-600 g, bought from Canadian Breeding Farm and Laboratories, St. Constant, Qu&ec. They were maintained in the 12-h light-dark197
0003-2697183 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reprcdutimn m any form reserved.
198
BRISSETTE
ness cycle and fed ad libiturn normal rat chow (Ralston Purina Co., St. Louis, MO.). Animals were anesthetized with diethyl ether for heart removal and with sodium pentobarbital (60 mg/kg body wt) for PHP collection. Lipoprotein lipase preparations. PHP was prepared as previously described (4). Acetone powder of heart tissue was prepared from overnight fasted rat hearts according to the method of Kom (5). Heparin-perfused rat heart LPL was obtained as follows: hearts were excised from fasted rats and the aorta was connected to a 161/z-gauge needle which was attached to the bottom opening of a three-way stopcock coupled to a 50-cm column filled with 0.15 M NaCl (saline) at room temperature. As soon as the heart was attached, the flow of saline from the column was immediately started. After 15 to 30 s of initial washing, 50 ml of ice-cold saline was perfused through the heart with a syringe fitted to the middle opening of the stopcock in order to carefully wash out most of the residual blood from the cardiac vascular space. Special care was taken to fill up the middle opening with saline from the column to remove all air bubbles before connecting the 50-ml syringe. After this extensive washing treatment, the hearts were slowly perfused using a syringe with 5 ml ice-cold saline containing 16 U of heparin and 10% glycerol (average flow rate of 1 ml/min). The LPLcontaining outflow was collected in a beaker kept on ice and reused to perfuse several washed hearts (up to 6) in order to enrich the preparation in lipase activity. Enzymatic assay. The enzyme activity of the LPL preparations was measured by using [9, 10-3H(N)]triolein (New England Nuclear, Boston, Mass., sp act 100-l 50 Ci/mmol) emulsion in glycerol as described by NilssonEhle and Schotz (19). The liberated free fatty acids were extracted (20) and counted in toluene-based scintillating solution with 40% efficiency. The enzymatic lipase activity of 1 mU was defined as the production of 1 nmol of oleic acid per minute at 37°C. Analytical determinations. The proteins
AND NOifL
were measured by the method of Lowry et al. (2 l), using bovine serum albumin as standard. Total lipids were extracted (22), separated by thin-layer chromatography, and determined as described (4). When needed, the LPL-containing solution was concentrated by using Aquacide II-A (Calbiochem-Behring Corp., La Jolla, Calif.). RESULTS
We have used the well-known property of heparin to release endothelium-bound LPL from perfused rat hearts (18). In order to get a LPL preparation with high specific activity, rat hearts were first extensively washed with 0.15 M NaCl to get rid of most of the serum proteins. Following this treatment, up to 6 hearts were perfused with the same heparin solution to enrich the preparation with LPL activity. We noted that the heparin solution gradually lost its ability to release cardiac LPL when more than 6 hearts were perfused with the same solution. If LPL from more than 6 hearts need to be prepared, more heparin should be added to the perfusate. This procedure was compared with two other rapid methods for preparing rat heart LPL. Table 1 shows that LPL from hearts perfused with a heparin solution containing 10% glycerol had a much higher specific activity than the rat heart acetone powder and the rat PHP. When glycerol was omitted from the heparin perfusate, the total LPL activity released from the heart was decreased 2.5-fold: 57.4 f 12.2 mU/g of heart with 10% glycerol versus 22.7 + 3.7 mU/g of heart without glycerol (mean + SEM, n = 3). The enzyme activity was also more stable when stored at 4°C if 10% glycerol was added to the perfusion medium. Figure 1 shows that in the absence of glycerol, the cardiac LPL lost 50% of its original activity after 3 days. In contrast, in the presence of 10% glycerol, only 25% of the LPL activity was lost during the first 2 days and it then remained stable for at least 8 more days. At 37”C, the glycerol preparation was also stable for 1 h (Table 2).
RAT TABLE
HEART
LIPOPROTEIN
TABLE
1
SPECIFICACTIVITY OF RAT LIPOPROTEIN LIPASE OBTAINED BY THREE DIFFERENT METHODS
Method
Specific activity” (mU/ mg protein)
Relative specific activity
99 *
34 (4)
1
378 f
28 (3)
4
4251 + 318 (3)
43
’ The results are the means f SEM of the number of experiments indicated in parentheses. The mU is defined as 1 nmol of free fatty acid liberated per minute at 37°C. ’ Fifty milligrams of acetone powder was dissolved in 1 ml of 25 mM ammonium hydroxide according to Kom (5). ‘The rat hearts were thoroughly washed with 50 ml of cold 0.15 M NaCl before perfusion with the heparin solution and perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol.
The activity, however, declined rapidly to less than 50% in the next 30 min. Even though the hearts were carefully perfused with 50 ml of saline, trace amounts of lipids could still be consistently detected in the LPL-containing perfusate, especially when several hearts were perfused sequentially with the same heparin solution. In order to evaluate the extent of lipid contaminants, the eluate
2
STABILITY OF THE PARTIALLY PURIFIED HEART LPL AT 37°C’ Time of incubation at 37°C (min) 0
Rat heart acetone powder b Rat postheparin plasma Heparin-perfused’ rat heart
199
LIPASE
Relative LPL activityb (% of zero time)
10
100.0 115.7 f
20 30 40 50 60
98.3 108.3 91.7 85.7 88.0
+ f + + +
3.2 (4)
13.9 (3) 13.5 (4) 7.5 (3) 14.1 (3) 14.0 (4)
a Washed hearts were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol. The LPL activity was measured by using a tritiated triolein emulsion as described under Materials and Methods. b The results are the means f SEM of the number of experiments indicated in parentheses.
from 10 perfused hearts was extracted and the lipids were separated by thin-layer chromatography. The mass of triacylglycerol, free and esterified cholesterol, as well as phospholipid, was determined. We calculated that each heart contributed approximately 6 to 9 nmol of each lipid class. Using electron microscopy at a magnification of 42,000 we failed to detect any lipoprotein particles in an enzyme preparation (from 10 hearts) concentrated 50 times. DISCUSSION
“0 8 *5’ t
I I I
FIG. 1. Stability of the perfused rat heart LPL at 4°C with (----) or without (- - -) 10% glycerol in the perfusion medium. Each point represents the mean of three determinations. Emymatic activity measured immediately after perfusion was taken as 100%.
The activity of purified rat heart LPL is particularly unstable at all temperatures. Ethylene glycol(1 M) appears to delay the decrease in enzyme activity at 4°C while glycerol (20%) somewhat stabilizes it at -20°C; the purified enzyme retained 56% of its initial activity after 7 days at -20°C (23). In this paper we present a partial characterization of a soluble rat heart LPL preparation which has both a relatively high specific activity and a reasonable stability. The observed stability at 37°C for 1 h is in agreement with the findings of Greten et al. (7), who have shown that PHP kept its lipase activity during 60 min incubation at 37°C. Like others, we observed that glycerol has
200
BRISSETTE
a stabilizing effect on the LPL activity at 4°C (Fig. 1). Furthermore, the presence of glycerol in the heparin perfusate increased 2.5fold the total enzyme activity releasable from the hearts. The concomitant release of nonspecific proteins was only increased 1.5-fold; therefore the specific activity of LPL was increased in the presence of glycerol. Glycerol appears to have two additive effxts: (a) it may promote the release of additional proteins (including LPL) from perfused rat hearts; (b) it may also stabilize the enzyme since the increment of nonspecific proteins was lower than that of total apparent LPL activity. Similarly, Crass and Meng (24) observed that the addition of 10% rat serum to the perfusate increased the release of total LPL activity from rat hearts. They ruled out the activation effect of apolipoprotein C-II since addition of 10% serum to the LPL solution (prepared in absence of serum) did not account for the observed increase. These authors concluded that serum appeared to favor the release of LPL from the heart tissue and not to activate or stabilize the enzyme. ACKNOWLEDGMENTS We thank Mireille Fyfe for assistance during heart perfusions and Marcel Smit for drawing the figure. The help of Rose-Mai Roy in typing the manuscript is greatly ap preciated. This work was supported by the Quebec Heart Foundation. L. Brissette is a recipient of a studentship from the Canadian Medical Research Council.
REFERENCES 1. Robinson, D. S. ( 1970) in Comprehensive Biochemistry (Florkin, M., and Stotz, E. W., eds.), Vol. 28, pp. 51-l 16, Elsevier, Amsterdam.
AND NOEL 2. Bilheimer, D. W., Eisenh S., and Levy, R. E. (1972) Biochim. Biophys. Acta 260, 212-221. 3. Eisenberg, S., and Rachmilewitz, D. (1975) J. Lipid Res. 16, 341-351. 4. Ndl, S-P., and Rubinstein, D. (1981) Canad. J. Biochem. 59,447-453. 5. Kom, E. D. (1955)J. Biol. Chem. 215, l-14. 6. Kom, E. D., and Quigley, T. W., Jr. (1957) J. Biol. Chem. 226, 833-838. 7. Greten, H., Levy, R. I., and Fredrickson, D. S. ( 1968) B&him. Biophys. Acta 164, 185-194. 8. Noel, S.-P., Dolphin, P. J., and Rubinstein, D. ( 1975) B&hem. Biophys. Res. Commun. 63, 764-77 1. 9. Chajek, T., and Eisenberg, S. (1978) J. Clin. Invest. 78, 1654-1665. 10. Pedersen, M. E., Wolf, L. E., and Schotz, M. C. ( 198 1) Biochim. Biophys. Acta 666, 191-199. 11. Fielding, C. J., and Higgins, J. M. (1974) Biochemistry 13,4324-4330. 12. Fielding, C. J. (1976) Biochemistry 15, 879-884. 13. Eisenbetg, S., Feldman, D., and Olivecrona, T. (198 1) B&him. Biophys. Acta 665,454-462. 14. Deckelbaum, R. J., Olivecrona, T., and Fainaru, M. (1980) J. Lipid Res. 21, 425-434. 15. Dory, L., Pocock, D., and Rubinstein, D. (1978) Biochim. Biophys. Acta 528, 161-175. 16. Tam, S. P., Dory, L., and Rubinstein, D. (1981) J. Lipid Res. 22, 64 l-65 1. 17. Ben-Zeev, O., Schwalb, H., and Schotz, M. C. (1981) J. Biol. Chem. 256, 10,550-10,554. 18. Fielding, C. J., and Higgins, J. M. (1974) Biochemistry 13,4324-4330. 19. Nilsson-Ehle, P., and Schotz, M. C. (1976) J. Lipid Res. 17, 536-541. 20. Trout, D. L., Estes, E. H., and Friedberg, S. J. ( 1960) J. Lipid Res. 1, 199-202. 21. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275. 22. Folch, L., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509. 23. Twu, J.-S., Garhnkel, A. S., and Schotz, M. C. (1975) Atherosclerosis 22, 463-472. 24. Crass, M. F. III, and Meng, H. C. (1964) Amer. J. Physiol. 206, 6 10-6 14.