ciprofibroyl-CoA synthetase was found in fat and liver homogenates. The distribution of ... are compatible with an identity of bothenzyme activities. These ... catalase for peroxisomes (Leighton et al., 1968); and. NADPH: ... 10 mm stock solution in 0.1 M-Tris/HCl buffer, pH 8.0, con- taining 0.5 ... The upper water/methanolic.
283
Biochem. J. (1992) 284, 283-287 (Printed in Great Britain)
Subcellular distribution and characteristics of ciprofibroyl-CoA synthetase in rat liver Its possible identity with long-chain acyl-CoA synthetase Ludwig AMIGO, Mary C. McELROY, M. Nelly MORALES and Miguel BRONFMAN* Faculty of Biological Sciences and Faculty of Medicine, Pontificia Universidad Cat6lica de Chile, Casilla 114-D, Santiago, Chile
The subcellular distribution and characteristics of ciprofibroyl-CoA synthetase were studied in rat liver and compared with those of long-chain acyl-CoA synthetase (palmitate as substrate) which, as already known, is distributed among mitochondria, microsomes and peroxisomes. Upon differential centrifugation, the subcellular distribution of ciprofibroylCoA synthetase followed closely that of palmitoyl-CoA synthetase and was specifically inactivated in the mitochondrial fraction by freezing and thawing, a behaviour already described for palmitoyl-CoA synthetase. -Both enzyme activities were found to co-purify through several steps from rat liver microsomes. By using a partially purified enzyme, the activation of ciprofibrate to its acyl-CoA ester followed Michaelis-Menten kinetics with an apparent Km of 0.63 + 0.1 mm. Ciprofibroyl-CoA synthetase was competitively inhibited by 25 and 50 /tM-palmitic acid. Higher concentrations of the fatty acid resulted in a mixed type of inhibition. Conversely, ciprofibrate up to 0.5 mm was found to inhibit competitively palmitoyl-CoA synthetase, whereas higher concentrations also resulted in a mixed inhibition. The highest activity of ciprofibroyl-CoA synthetase was found in fat and liver homogenates. The distribution of the enzyme in different rat tissues was similar to that of palmitoyl-CoA synthetase. The present results suggest that long-chain acyl-CoA synthetase and ciprofibroyl-CoA synthetase activities reside in identical or closely related proteins.
INTRODUCTION We have previously reported that three hypolipidaemic drugs of the fibrate series undergo activation to acyl-CoA thioesters catalysed by rat liver microsomes (Bronfman et al., 1986). Other hypolipidaemic compounds, non-structurally related to fibrates, such as Medica 16 and bis(carboxymethylthio)- 1, 10-decane, are similarly activated to acyl-CoA esters by rat liver microsomes (Hertz & Bar Tana, 1988; Aarsland et al., 1990). All these compounds are known to cause hepatomegaly in treated animals, together with peroxisomal proliferation and other important metabolic effects [for a review, see Hawkins et al. (1987)]. In addition, they have been proposed as a new class of chemical carcinogens in rat and mice (Reddy et al., 1980). The multiple effects induced by hypolipidaemic peroxisome proliferators in the liver might be related to the formation of their acyl-CoA esters, since the metabolites of these drugs couldaffect all metabolic pathways involving acyl-CoAs, as either substrates or modulators (Bronfman et al., 1986). In addition, CoA esters of five of these carcinogenic peroxisome proliferators have been shown to potentiate the activity of protein kinase C (Bronfman et al., 1989; Orellana et al., 1990), a kinase involved in cell differentiation and tumour promotion (Nishizuka, 1984). These hypolipidaemic compounds have been also shown to acylate membrane and cytosolic proteins in cultured hepatocytes (Hertz & Bar Tana, 1988). The enzyme activity responsible for the activation of hypolipidaemic peroxisome proliferators has not been the subject of a comprehensive study. It is not known by which enzyme the reaction is catalysed, and its subcellular localization has not been determined. Several subcellular organelles might be potentially able to activate these drugs. Long-chain-fatty-acyl-CoA synthetase (fatty acid: CoA ligase, EC 6.2.1.3), th-e enzyme responsible for activat*
To whom correspondence should be addressed.
Vol. 284
ing most natural fatty acids, is localized in microsomes, in the outer membrane of mitochondria and in peroxisomes (Krisans et al., 1980; Mannaerts et al., 1982; Bronfman et al., 1984). The enzyme purified from these organelles is apparently the same protein (Tanaka et al., 1979; Miyazawa et al., 1985), although a distinct very-long-chain-fatty-acyl-CoA synthetase has been reported in rat liver microsomes and peroxisomes (Singh & Poulos, 1988). Cholic acid and trihydroxycoprostanic acid are also activated to acyl-CoA thioesters by separate enzymes (Schepers et al., 1989), whereas prostaglandins and dicarboxylic acids are activated solely in the endoplasmic reticulum (Vamecq et al., 1985; Schepers et al., 1988). In the present work we studied the subcellular distribution of the acyl-CoA synthetase responsible for the activation of hypolipidaemic peroxisome proliferators, using ciprofibrate as a model compound. We have compared the characteristics of the enzyme with those of long-chain-acyl-CoA synthetase and determined its tissue distribution. Our results show that ciprofibroyl-CoA synthetase has the same subcellular distribution as long-chainacyl-CoA synthetase and the same inactivation pattern in mitochondrial fractions. Both enzyme activities co-purify and have similar tissue distributions. Furthermore, their kinetic properties are compatible with an identity of both enzyme activities. These observations suggest that ciprofibrate is activated by long-chainacyl-CoA synthetase with a multiple subcellular localization. EXPERIMENTAL Materials
Ciprofibrate was kindly provided by Sterling-Winthrop Research Institute, Rensselaer, NY, U.S.A. Coenzymes, Triton X-100, substrates and chemicals were from Sigma Chemical Co., St. Louis, MO, U.S.A. H.p.l.c.-grade methanol was purchased from Merck, Darmstadt, Germany. [1-14C]Palmitate (sp. radio-
284 activity 56 mCi/mmol) was obtained from New England Nuclear, Boston, MA, U.S.A. Animals
Sprague-Dawley rats, weighing 180-200 g, were used. They maintained on a standard laboratory chow diet.
were
Preparation of subcellular fractions Tissue homogenates (20 %, w/v) were prepared in 0.25 Msucrose containing 1 mM-dithiothreitol and 3 mM-imidazole/ HCI, pH 7.4. Liver homogenates were fractionated into nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsoma! (P) and soluble (S) fractions, as described by de Duve et al. (1955). For palmitoyl-CoA synthetase purification, the N and M fractions were sedimented together (NM fraction), and the microsomal fraction was directly sedimented from the NM-fraction supernatant. This microsomal fraction contained 60-70 % of the homogenate palmitoyl-CoA synthetase.
Determination of enzymes and protein Standard procedures were used for the determination of marker enzymes: glutamate dehydrogenase for mitochondria; catalase for peroxisomes (Leighton et al., 1968); and NADPH: cytochrome c reductase for microsomal vesicles (Beaufay et al., 1974). Protein was measured by the method of Lowry et al. (1951), with BSA as standard. Palmitoyl-CoA synthetase was assayed by the radiochemical assay described by Krisans et al. (1980). The incubation mixture contained 5 mM-ATP, 5 mM-MgCl2, 2 mM-CoA, 1 mM-dithiothreitol, 0.1 M-Tris/HCI buffer, pH 8.0, 0.15 % Triton X-100 and 0.4 mM-[1-_4C]palmitate (0.5 ,uCi/ml) in a total volume of 0.2 ml. Incubation was performed for 10 min at 37 'C. The palmitoylCoA formed was quantified as described by Krisans et al. (1980). Ciprofibroyl-CoA synthetase was measured using the same reaction mixture and total volume as for palmitoyl-CoA synthetase (Bronfman et al., 1986). The drug was prepared as a 10 mm stock solution in 0.1 M-Tris/HCl buffer, pH 8.0, containing 0.5 % Triton X-100. The final concentration of the drug in the reaction mixtures was 3 mm. After incubation at 37 'C for 30 min, the reaction was stopped by the addition of 0.8 ml of chloroform/methanol (2:1, v/v). The upper water/methanolic phase was used directly for the analysis of ciprofibroyl-CoA by h.p.l.c. In previous experiments, using chemically synthesized ciprofibroyl-CoA (Bronfman et al., 1989), it has been determined that more than 95 % of the ciprofibroyl-CoA was recovered in the upper phase under these conditions. H.p.l.c. separation was performed on an RP- 18 (5 ,sm) column (LichroCart 125-4; Merck, Darmstadt, Germany), using as mobile phase 0.5 MKH2PO4, pH 5.0/water/methanol (1:4: 5, by vol.) at 1.5 ml/min. Under these conditions, ciprofibrate and ciprofibroyl-CoA retention times were 6.8 and 12 min respectively. Ciprofibroyl-CoA was estimated by spectrophotometric detection and recording at 254 nm. Authentic ciprofibroyl-CoA was used as standard. The reaction was linear with respect both to time (up to 45 min incubation) and to protein concentration (up to 0.1 mg of protein per assay) with either total homogenates or subcellular fractions. Purification of palmitoyl-CoA synthetase For palmitoyl-CoA synthetase purification, rat liver microsomes prepared as described above were extracted with 0.15 % Triton X-100 by following the general procedure described by Merril et al. (1982). Further purification of the enzyme by Blue Sepharose and phosphocellulose chromatography was performed by the method of Tanaka et al. (1979). In brief, Triton X-100solubilized proteins, isolated from 20 g of liver, were loaded on
L. Amigo and others 1.5 cm x 15 cm Blue Sepharose column equilibrated in 20 mmKH2PO4 buffer, pH 7.4, containing 2 mM-Triton X-100 and 5 mM-dithiothreitol (buffer A). The column was washed with 50 ml of buffer A, followed by 50 ml of buffer A containing 10 mM-ATP. Acyl-CoA synthetase was eluted with 50 ml of buffer A containing 5 mM-ATP and 1 M-NaCl. Fractions with enzyme activity were pooled (48 ml), and a sample of the pooled fractions (14 ml; 3.2 mg of protein) was dialysed for 6 h in buffer A and loaded on to a phosphocellulose column (2.6 cm x 1 cm) equilibrated with buffer A. After washing with 10 ml of buffer A, enzyme activities were eluted with a linear gradient (80 ml total volume) from buffer A to 0.35 M-KH2PO4 in buffer A. Fractions (4 ml each) were collected.
a
RESULTS Subcellular distribution The subcellular distribution of ciprofibroyl-CoA synthetase and palmitoyl-CoA synthetase, together with the distribution of marker enzymes, are shown in Fig. 1. Two distribution patterns are presented for the synthetases. One was obtained after directly assaying the activities on the fresh fractions, and the other after freezing and thawing the fractions before the assay. When the activity was measured in fresh fractions, palmitoyl-CoA synthetase showed a microsomal-mitochondrial pattern of distribution, consistent with previously published distributions in rat (Krisans et al., 1980) and human liver (Bronfman et al., 1984).
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Fig. 1. Subceliular distribution of ciprofibroyl-CoA synthetase and palmitoyl-CoA synthetase Rat liver homogenates were fractionated by differential centrifugation into nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and soluble (S) fractions, and enzymes were measured in each fraction. (a) Palmitoyl-CoA synthetase and (b) ciprofibroyl-CoA synthetase, measured in fresh fractions. (c) Palmitoyl-CoA synthetase and (d) ciprofibroyl-CoA synthetase measured after freezing and thawing the fractions. Marker enzymes: (e) catalase (peroxisomal matrix); (f) NADPH:cytochrome c reductase (microsomes); (g) glutamate dehydrogenase (mitochondrial matrix). Results are expressed as relative specific activities versus percentage of total protein. Relative specific activity is defined as the percentage of total recovered activity present in a particular fraction divided by the corresponding percentage of protein. Recoveries ranged between 80 and 110 %. Total homogenate activities of ciprofibroyl-CoA synthetase and palmitoyl-CoA synthetase decreased 35 and 40 % respectively after freezing and thawing, whereas no significant changes were observed for the distribution or total activity of marker enzymes. Essentially similar results were obtained in two independent experiments.
1992
285
Characteristics of ciprofibroyl-CoA synthetase
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Fig. 2. Phosphoceliulose chromatography of acyl-CoA synthetase Fractions obtained from Blue Sepharose chromatography of Triton X- 100-solubilized microsomal proteins were loaded on to a phosphocellulose column as described in the Experimental section. The column was washed with equilibration buffer, and eluted with a linear gradient (arrow) from 20 mM- to 0.35 M-KH2PO4 in equilibration buffer. Palmitoyl-CoA synthetase (O), ciprofibroylCoA synthetase (0) and protein (A) were measured in each fraction.
When assayed after freezing and thawing the fractions, palmitoylCoA synthetase distribution changed into a microsomal-like pattern, resulting from inactivation of the enzyme in the mitochondrial fractions (Tanaka et al., 1979). Ciprofibroyl-CoA synthetase distribution closely follows the pattern of palmitoylCoA synthetase when assayed in fresh fractions or after freezing and thawing the fractions before the assay. These results suggest that the non-specific long-chain-acyl-CoA synthetase with multiple subcellular localization might be responsible for ciprofibrate activation. Partial purification of ciprofibroyl-CoA synthetase To obtain further evidence for the possible identity of ciprofibroyl-CoA synthetase and palmitoyl-CoA synthetase, rat liver microsomes were extracted with Triton X-100 and the supernatant was subjected Blue Sepharose chromatography as de-
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[Palmitatel (mM) [Ciprofibratel (mM) Fig. 3. Effects of pahnitate on ciprofibroyl-CoA synthetase and of ciprofibrate on palmitoyl-CoA synthetase The activation of ciprofibrate at increasing concentrations of palmitate (a) and that of palmitate at increasing concentrations of ciprofibrate (b) were measured in the Blue Sepharose-purified acylCoA synthetase as described in the Experimental section. In both cases results are presented as percentages of the activity in the absence of palmitate or ciprofibrate respectively. Similar results were obtained for (a) and (b) in two and three independent experiments respectively. Palmitoyl-CoA synthetase inhibition by ciprofibrate was statistically significant at 0.5 mm and higher concentrations of the drug.
scribed above. In both steps, enzyme activities using either palmitate or ciprofibrate were found to co-purify. Further purification of the enzyme using phosphocellulose chromatography also resulted in co-elution of both enzyme activities (Fig. 2). As shown in Table 1, a similar increase was found in the specific activity of acyl-CoA synthetase relative to the initial homogenate, using either palmitate or ciprofibrate as substrate throughout the purification steps.
Substrate-competition experiments By using the Blue Sepharose-purified enzyme, palmitate was found to inhibit strongly ciprofibroyl-CoA synthetase (Fig. 3a). Almost 75 % inhibition was found at 0.3 mm of the fatty acid. Inversely, ciprofibrate was found to inhibit palmitoyl-CoA synthetase (Fig. 3b), although higher concentrations of the drug were needed to demonstrate this effect, and the maximal inhibition obtained was only 20-30 %. Kinetic studies of both enzyme activities were carried out. Michaelis-Menten kinetics, with an apparent Km of
Table 1. Purification of acyl-CoA synthetase Acyl-CoA synthetase was purified from rat liver as described in the Experimental section. Activity was assayed using palmitate or ciprofibrate as substrate. Relative specific activities (RSA) in each fraction are presented in relation to that in the initial homogenate. Total recovered activities in the phosphocellulose fraction were multiplied by a factor of 3.4, since only 29 % of the Blue Sepharose fraction was applied to the phosphocellulose column (see the Experimental section).
Palmitoyl-CoA synthetase Total activity
Fraction
(mg)
(,umol/min)
(%)
RSA
(,umol/min)
(%)
RSA
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27.3 34.5
0.34 0.12
Microsomal fraction Triton X-100-solubilized protein fraction Blue Sepharose fraction Phosphocellulose fraction
Vol. 284
Ciprofibroyl-CoA synthetase
Total protein
11 2.7
8.4 2.6
Total activity
Yield
7.6 2.4
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68 42 6.6 2.3
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[Ciprofibratel (mm) Fig. 4. Effect of palmitate on ciprofibroyl-CoA synthetase The activation of ciprofibrate was measured in the Blue Sepharosepurified acyl-CoA synthetase at increasing substrate concentrations, in the absence (0) and in the presence of 25 /,M (0), 50/M (C1) and 300 ,uM (E)-palmitate. Results are plotted as rates against concentrations. Apparent Km values of 0.63 +0.1, 1.02+0.16, 1.30+0.08 and 2.80 + 0.40 mm in the presence of 0, 25, 50 and 300 ,iM-palmitate respectively were calculated from the data by direct fitting to a rectangular hyperbola using a standard computer program. Vmax values of 52.0 + 3.0, 54.0 + 3.6, 51.8 + 1.48 and 20.0 + 1.78 nmol/min per mg of protein in the presence of 0, 25, 50 and 300 ,uM-palmitate were determined using the same program. Double-reciprocal linear transformations of the curve in the presence of 300 ,uM-palmitate are presented in inset (a). Inset (b) shows double-reciprocal linear transformations of the curves in the presence of 0 (O), 25 (M) and 50 (El) /LM-palmitate. The dotted line in inset (b) shows the doublereciprocal linear transformation of the curve in the presence of 300 /LM-palmitate shown in (a), projected to the scale in (b).
0
0.2
0.1
0.3
[Palmitatel (mm)
Fig. 5. Effect of ciprofibrate on palmitoyl-CoA synthetase The activation of ciprofibrate was measured with the Blue Sepharosepurified acyl-CoA synthetase at increasing substrate concentrations, in the absence (O) and in the presence of 0.5 (-) and 1.5 (rO) mmpalmitate. Results are plotted as rates against concentrations. Apparent Km values of 21. 1 + 5.3, 29.5 + 6.5 and 40.2 + 7.3 #m in the presence of 0, 0.5 and 1.5 mm-ciprofibrate respectively were calculated from the data by direct fitting to a rectangular hyperbola using a standard computer program. Vm..' values were 0.85 +0.04, 0.84+0.05 and 0.77+0.0410mol/min per mg of protein in the m-iprofibrate respectively. Doublepresence of 0, 0.5 and 1.5m
reciproval linear transformations of the curves are presented in the inset (same symbols).
0.63+0.1 mM, were observed when using ciprofibrate as substrate. Low concentrations of palmitate competitively inhibited ciprofibroyl-CoA synthetase, whereas a mixed inhibition was found at higher concentrations of the fatty acid (Fig. 4). When palmitate was used as substrate, Michaelis-Menten kinetics was also observed, with an apparent Km of about 20 ,m, which is in good agreement with previously reported values (Tanaka et al., 1979). Inhibition studies of palmitoyl-CoA synthetase by ciprofibrate were complicated by the low inhibitory action of the drug, probably resulting from the almost 30-fold difference between the respective Km values. However, it was consistently observed that ciprofibrate up to 0.5 mm competitively inhibited palmitoyl-CoA synthetase, whereas higher concentrations resulted in a mixed inhibition, as in the case of ciprofibroyl-CoA synthetase inhibition by high palmitate concentrations. One of these experiments is presented in Fig. 5. Tissue distribution Synthetase activities were measured in homogenates from several rat tissues. As shown in Table 2, palmitoyl- and ciprofibroyl-CoA synthetases had a similar tissue distribution. The highest activity occurred in fat and liver. It is noteworthy that a good correlation exists between the activity of palmitoyl-CoA and that of ciprofibroyl-CoA synthetase in the different tissues (the correlation coefficient, calculated from Table 2, was 0.989).
DISCUSSION The results presented here show that palmitoyl-CoA and
Table 2. Tissue distribution of palmitoyl-CoA synthetase and ciprofibroylCoA synthetase Activities were determined in total homogenates as described in the Experimental section. Results are means + S.E.M. for separate results obtained with three rats.
Enzyme activity (nmol/min per mg of protein) Tissue
Fat (abdominal) Liver Fat (epididymal) Heart Intestinal mucosa
Kidney
Palmitoyl-CoA synthetase
Ciprofibroyl-CoA synthetase
50.64+ 19.18 33.01 + 7.36 31.33 + 12.26 7.22 + 1.52 7.05 + 1.05 3.71 + 1.00
2.42+ 1.32 1.60+0.39 1.21+0.44 0.34+0.22 0.43 +0.31 0.07 + 0.05
ciprofibroyl-CoA synthetases display identical subcellular distribution and have a similar inactivation pattern in mitochondrial fractions. Both enzymes co-purify from microsomal fractions and present a similar tissue distribution. In addition, ciprofibrate and palmitate at low concentrations were found to inhibit competitively palmitoyl-CoA and ciprofibroyl-CoA synthetases respectively. All these observations are compatible with the identity of both enzyme activities and suggest that ciprofibrate, 1992
287
Characteristics of ciprofibroyl-CoA synthetase and probably other related compounds, are activated by the nonspecific long-chain-acyl-CoA synthetase. Further studies on subcellular distribution and characteristics of the enzyme activating other carboxylic-group-containing hypolipidaemic peroxisome proliferators could help to elucidate this hypothesis. The mixed inhibition observed at high concentrations by either ciprofibrate or palmitate towards palmitoyl-CoA and ciprofibroyl-CoA synthetases respectively might result from the detergent properties of these molecules. Mixed or competitive inhibition has not been observed in the case of the related enzymes choloyl-CoA synthetase, trihydroxycoprostanoyl-CoA synthetase and palmitoyl-CoA synthetase, which are separate enzymes (Schepers et al., 1989). In this case palmitate inhibits non-competitively both the cholic and trihydroxycoprostanic activating enzyme, whereas bile acids do not inhibit palmitoylCoA synthetase. The characterization of the enzyme activating hypolipidaemic peroxisome proliferators is of importance, since on the basis that these drugs are a rather heterogeneous group of compounds but produce similar metabolic effects in the liver, it has been proposed that their acyl-CoA derivatives might be the common pharmacologically active species of these compounds (Bronfman et al., 1986; Aarsland et al., 1990). Long-chain-acyl-CoA synthetase has a multiple subcellular localization. Thus if hypolipidaemic compounds are indeed activated by this enzyme, the generation of their CoA esters might also have a multiple subcellular localization. It is noteworthy that hypolipidaemic compounds affect not only peroxisomes. Mitochondrial f-oxidation, microsomal w-oxidation and several soluble proteins related to acylCoA metabolism or transport are also affected (Hawkins et al., 1987). The possible multiple subcellular localization of CoA ester synthesis of these drugs may facilitate their action on specific organelles. It has been shown that bezafibrate, an analogue of ciprofibrate, inhibits fatty acid oxidation in isolated hepatocytes, whereas bezafibroyl-CoA inhibits overt carnitine palmitoyltransferase I in rat liver mitochondria (Eacho & Foxworthy, 1988). The access of the CoA esters of the drugs to intraorganelle spaces is also matter of interest. Van Veldhoven et al. (1987) have shown that peroxisomes are permeable to several molecules, including ATP, CoA and NADI. Using the techniques of these authors, we have found that apparently intact peroxisomes are permeable to the CoA ester of nafenopin, an analogue of ciprofibrate, and that this ester strongly inhibits the peroxisomal fatty-acyl-CoA-oxidizing system (G. Loyola & M. Bronfman, unpublished work). Received 7 August 1991/31 October 1991; accepted 11 November 1991
Vol. 284
This work was supported by the Fondo Nacional de Investigaci6n (Fondecyt 802/90). We thank Dr. Manuel Santos, Dr. Octavio Monasterio and Dr. Tito Ureta for helpful discussion and suggestions.
REFERENCES Aarsland, A., Berge, R. K., Bremer, J. & Aarsaether, N. (1990) Biochim. Biophys. Acta 1033, 176-183 Beaufay, H., Amar-Costesec, A., Feytmans, E., Thines-Sempoux, D., Wibo, M., Robbi, M. & Berthet, J. (1974) J. Cell Biol. 61, 188-200 Bronfman, M., Inestrosa, N. C., Nervi, F. 0. & Leighton, F. (1984) Biochem. J. 224, 709-720 Bronfman, M., Amigo, L. & Morales, M. N. (1986) Biochem. J. 239, 781-784 Bronfman, M., Orellana, A., Morales, M. N., Bieri, F., Waechter, F., Staubli, W. & Bentley, P. (1989) Biochim. Biophys. Res. Commun. 159, 1026-1031 de Duve, C., Pressmann, B. C., Giannetto, R., Wattiaux, R. & Appelmans, F. (1955) Biochem. J. 60, 604-617 Eacho, P. I. & Foxworthy, P. S. (1988) Biochem. Biophys. Res. Commun. 157, 1148-1151 Hawkins, J. M., Jones, W. E., Bonner, F. W. & Gibson, G. G. (1987) Drug Metab. Rev. 18, 441-515 Hertz, R. & Bar-Tana, J. (1988) Biochem. J. 254, 39-44 Krisans, S. K., Mortensen, R. M. & Lazarow, P. B. (1980) J. Biol. Chem. 255, 9599-9607 Leighton, F., Poole, B.., Beaufay, H., Baudhuin, P., Coffey, J. W., Fowler, S. & de Duve, C. (1968) J. Cell Biol. 37, 482-513 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Mannaerts, G. P., Van Veldhoven, P., Van Broekhoven, A., Vandebroek, G. & Debeer, L. K. (1982) Biochem. J. 204, 17-23 Merril, A. H., Gidwitz, S. & Bell, R. M. (1982) J. Lipid Res. 23, 1368-1372 Miyazawa, S., Hashimoto, T. & Yokota, S. (1985) J. Biochem. (Tokyo) 98, 723-733 Nishizuka, Y. (1984) Nature (London) 308, 693-697 Orellana, A., Hidalgo, P., Morales, M. N., Mezzano, D. & Bronfman, M. (1990) Eur. J. Biochem. 190, 57-61 Reddy, J. K., Azarnoff, D. L. & Hignite, C. E. (1980) Nature (London) 283, 397-398 Schepers, L., Casteels, M., Vamecq, J., Parmentier, G., Van Veldhoven, P. P. & Mannaerts, G. P. (1988) J. Biol. Chem. 263, 2724-2731 Schepers, L., Casteels, M., Verheyden, K., Parmentier, G., Asselberghs, S., Eyssen, H. J. & Mannaerts, G. P. (1989) Biochem. J. 257, 221-229 Singh, H. & Poulos, A. (1988) Arch. Biochem. Biophys. 266, 486-495 Tanaka, T., Hosaka, K., Hoshimaru, M. & Numa, S. (1979) Eur. J. Biochem. 98, 165-172 Vamecq, J., De Hoffmann, E. & Van Hoff, F. (1985) Biochem. J. 230, 683-693 Van Veldhoven, P. P., Just, W. W. & Mannaerts, G. P. (1987) J. Biol. Chem. 262, 4310-4318
