NOTES
Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol SHOBHA GHOSHAND W. MCLEANGROGAN'
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Department of Biochemistry and Molecular Biophysics, Box 614, MCV Station, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0614, U.S.A. Received March 17, 1992
GHOSH, S., and GROGAN, W.M. 1992. Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol. Biochem. Cell Biol. 70: 800-803. Rabbit polyclonal antibodies were raised against rat liver bile salt-independent neutral cholesteryl ester hydrolase (CEH) and used for subcellular localization and immunological comparison with isoforms from other tissues. Antibodies exhibited a high degree of specificity for the liver CEH through all stages of purification and neutralized 70-80% of the activity of liver cytosolic CEH. They exhibited various levels of cross-reactivity with cytosolic proteins from other tissues, but reacted weakly with pancreatic and intestinal proteins and did not inhibit pancreatic CEH. Cytosol contained 78% of total cellular CEH activity and 75% of CEH immunoreactive protein. Washed microsomes contained 3% of CEH activity and 5% of CEH protein. Key words: cholesteryl esterase, polyclonal antibodies, rat liver, subcellular distribution.
GHOSH, S., et GROGAN, W.M. 1992. Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol. Biochem. Cell Biol. 70 : 800-803. Des anticorps polyclonaux de lapin sont ClevCs contre la cholesteryl ester hydrolase (CEH) neutre independante des sels biliaires de foie de rat et utilises pour la localisation subcellulaireet la comparaison immunologique avec les isoformes des autres tissus. Les anticorps manifestent un haut degre de specificit6 pour la CEH hepatique a tous les stades de la purification et ils neutralisent 70-80% de l'activite de la CEH cytosolique hkpatique. 11s exhibent divers taux de rkactivite avec les prottines cytosoliques d'autres tissus, mais ils reagissent faiblement avec les protkines pancrkatiques et intestinales et ils n'inhibent pas la CEH pancreatique. Le cytosol contient 78% de l'activite cellulaire totale de la CEH et 75% de la protkine CEH immunorCactive. Les microsomes renferment 3% de I'activitC CEH et 5% de la protiine CEH. Mots clks : cholesteryl esttrase, anticorps polyclonaux, foie de rat, distribution subcellulaire. [Traduit par la redaction]
Introduction Neutral cholesteryl ester hydrolases occur in many tissues, including liver (Deykin and Goodman 1962), pancreas (Calame et al. 1975), adrenals (Trzeciak and Boyd 1974), gonads (Durham and Grogan 1982; Behrman and Greep 1972), mammary glands (Martinez and Botham 1990), brain (Eto and Suzuki 1972), and corpus luteum (Cook et al. 1983), where they catalyze release of free cholesterol from cholesteryl ester, an intracellular storage form of cholesterol. Hepatic CEH, along with HMG-CoA reductase, cholesterol 7a-hydroxylase, and acyl-CoA:cholesterol acyltransferase, is a potentially important enzyme in regulation of the bile acid precursor (free cholesterol) pool in the liver. Multiple forms of CEH have been reported in testis (Durham and Grogan 1984), adrenals (Pittman and Steinberg 1977), and brain (Eto and Suzuki 1972), as well as liver (Deykin and Goodman 1962; Nilsson 1976). We have previously reported ABBREVIATIONS: CEH, cholesteryl ester hydrolase: HMG-CoA, TEMED, hydroxymethyl glutaryl coenzyme A; PI, isoeiectric N,N,N1, Nf-tetramethylethylenediamine; IgG, immunoglobulin G; PVDF, polyvinylidene difluoride; PEG, polyethylene glycol 8000; ABTS, 2,2'-azino-bis(3-ethylbenzthiazone-6-sulfonic acid); SDS, sodium dodecyl sulfate; kDa, kilodalton(s); CFA, complete Freund's adjuvant; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PBS, 100 mM phosphate buffer (pH 7.5) containing 150 mM NaCl. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
purification and characterization of a protein kinase activated CEH from rat liver cytosol, which did not require bile salt for activity (Ghosh and Grogan 1991). This CEH differed from pancreatic CEH with respect to bile salt dependence, subunit behavior, and PI, and did not crossreact with antibodies raised against rat pancreatic CEH (Ghosh and Grogan 1991). Although neutral bile saltindependent CEH activity has been reported in both cytosolic and particulate fractions from rat liver (Deykin and Goodman 1962; Nilsson 1976), the relationship between these activities has not been clarified. To characterize neutral hepatic CEH immunologically and determine its subcellular distribution, we have produced polyclonal antibodies to the purified enzyme. Using these antibodies we demonstrate that this enzyme is primarily cytosolic and accounts for at least 78% of total CEH activity in rat liver. Immunological comparison between enzymes from different organs is also described. Materials and methods Cholesteryl [l-'4~]oleate(56.6 mCi/mmol) was purchased from New England Nuclear (Boston, Mass.). All solvents were purchased from Fischer Scientific (Columbia, Md.). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, goat anti-rabbit IgG horse radish peroxidase conjugate, diaminobenzidine, and dye binding reagent for protein estimation were obtained from Bio-Rad Laboratories (Richmond, Calif.). Immobilon PVDF transfer mem-
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FIG. 1. Western blot analysis of rat liver neutral cytosolic CEH at different stages of purification. Proteins were separated by SDSPAGE and Western blots were developed as described in the Materials and methods. (A) Twenty-five micrograms of cytosolic protein; (B) 25 pg protein from 0 to 40% ammonium sulfate precipitate; (C) pooled and concentrated fractions from Mono S column, 12 pg protein; (D) concentrated fraction from gel permeation column, 2.7 pg protein (pure CEH).
branes were from Millipore (Bedford, Mass.). PEG, ammonium sulfate, ABTS, and Trizma base were purchased from Sigma Chemical Co. (St. Louis, Mo.). All other chemicals used were of analytical grade. Production of polyclonal antibodies CEH was purified from rat liver cytosol as described earlier (Ghosh and Grogan 1991). The pooled and concentrated fraction from the last step of purification (approximately 50-60 pg protein) was subjected to 5-20% SDS-polyacrylamide gel electrophoresis and the region corresponding to 66 kDa was cut and used for immunization. For the primary immunization, gel pieces were homogenized in saline and CFA (gel-saline-CFA, 3:1:4) and injected intradermally at 20-25 sites on the back of New Zealand rabbit on day 0. Subsequent booster immunizations were performed on day 14 and day 28 using the same amount of protein in gel pieces homogenized in incomplete adjuvant. The rabbit was test bled 7 days after the second booster and serum tested for antibodies using Western analysis as described below. A booster was given in incomplete adjuvant 7 days prior to subsequent bleedings. IgG was prepared from the test serum and preimmune serum (collected 7 days prior to immunization) by PEG precipitation as described by Carter and Boyd (1979). Subcellular fractionation Subcellular fractions were prepared from rat liver homogenate by differential centrifugation, as described earlier (Ghosh and Grogan 1989). All particulate fractions were washed once with the homogenizing buffer and resuspended in the same buffer for CEH assay and Western blot analysis. Western blot analysis Proteins separated by SDS-PAGE were electroblotted onto Immobilon-PVDF membrane using Integrated Separation System's semi-dry electroblotter. Western blots were developed following the standard protocol, using 5% nonfat dry milk in TBS (20 mM Tris-HC1 (pH 7.5) containing 150 mM NaCl) as the blocking solution, 1:200 dilution of immune serum in 1Yo gelatin in TBS as primary antibody, goat anti-rabbit IgG horse radish peroxidase conjugate as secondary antibody, and diaminobenzidine .as the color reagent. Western blots were scanned at 350 nm using a Shimadzu densitometer.
pg IgG Added FIG. 2. Neutralization of CEH activity in cytosol with anti-rat liver CEH IgG. Cytosolic protein (500-600 pg) was preincubated or immune ( a , anti-liver CEH) with 10-160 pg of preimmune (0) IgG and CEH activity was measured as described in Materials and methods. Data is plotted as percent CEH activity remaining, where 100% control was 2.3 + 0.1 nmol/h. Values are mean SEM of duplicates. Experiment was repeated with similar results.
*
ELISA Increasing concentrations of purified CEH protein were used to coat the plates and ELISA was carried out as described by Voller et al. (1979). ABTS was used as the color reagent. Neutralization assay Cytosolic protein (500-600 pg) was incubated with increasing concentrations of either preimmune or immune IgG (in PBS) at room temperature with vigorous shaking for 30 min. Following incubation, the volume was brought to 500 pL with 20 mM TrisHCl buffer (pH 7.5) containing 5 mM 2-mercaptoethanol and CEH activity was measured as described earlier (Ghosh and Grogan 1989). Protein determination Protein was measured by the Bio-Rad dye binding assay according to the manufacturer's instructions.
Results and discussion Specificity of antiserum Antiserum to liver CEH was tested for antibodies directed against neutral CEH from rat liver, by Western blot analysis. A positive reaction was observed with purified CEH from rat liver (Fig. ID). No reaction was observed with bovine serum albumin as a nonspecific control (data not shown). Antiserum also reacted with only one major protein band in cytosol and fractions representing different stages of purification of CEH (Fig. 1 , A-C). This band corresponded to a molecular mass of 66 kDa, the molecular mass of CEH (Ghosh and Grogan 1991). One or more minor bands were sometimes observed in cytosol and ammonium sulfate precipitate, the number and intensity of which increased upon storage, suggesting proteolytic degradation. These results confirm specificity of the polyclonal antibodies raised against CEH and suggest that this enzyme is immunologically different from other proteins in rat liver cytosol.
BIOCHEM. CELL BIOL. VOL. 70, 1992
802
TABLE 1. Subcellular distribution of liver neutral CEH
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-
Fraction
% total activity
% total CEH protein
Cytosol Unwashed microsomes Washed microsomes Crude mitochondria1 fraction
7k2.1
11k0.8
k D a 1 2 3 4
5 6 7 8
CEH activity and immunoreactive protein were measured in cytosolic and particulate fractions obtained from rat liver by differential centrifugation, as described in the Materials and methods. Data are presented as percent total liver activity, which was 391 nmol/h. CEH protein was measured by Western blot analysis, followed by densitomeric scanning. Values represent mean + SEM of two experiments in duplicate.
Specificity was also confirmed using ELISA. A positive linear correlation was observed between protein concentration of pure CEH and absorbance at 405 nm (r = 0.93) in a standard ELISA (data not shown). Neutralization of enzyme activity To confirm that antibodies were directed against CEH and to determine the fraction of activity represented by the CEH which we have purified, the capacity of antibodies to inhibit cytosolic CEH activity was examined. Whereas serum alone was found to inhibit CEH (S. Ghosh and W.M. Grogan, unpublished results), purified immune and preimmune control IgG were used in this study. Incubation with increasing concentrations of immune IgG resulted in increasing inhibition of CEH activity relative to preimmune control IgG (Fig. 2). Inhibition by 70-80% was observed at the highest IgG concentration used, strongly suggesting that the enzyme purified by us is the major cytosolic CEH in rat liver. In a separate experiment, pancreatic CEH was not significantly inhibited (81 k 5070, activity remaining, versus 37 10070,activity remaining in inhibited liver cytosol) by a single concentration of anti-liver CEH IgG (100 pg). These results support conclusions in our recent report (Ghosh and Grogan 1991) in which, based on high yield and specific activity, we suggested that this CEH is the predominant activity in rat liver cytosol. Some inhibition was also observed with preimmune IgG (Fig. 2). This is similar to the inhibition of other enzymes by immunoglobulins reported by others and attributed to nonspecific interaction of immunoglobulins with unstable enzymes (Schimke 1975). Subcellular distribution of CEH in rat liver There has been considerable confusion and some controversy concerning the relative contributions of cytosolic and particulate forms of CEH in the liver. We have addressed this question directly by measuring CEH activity and enzyme mass in liver cytosol, the crude mitochondrial fraction, and microsomes. As seen in Table 1,78% of total CEH activity and 75% of CEH protein were associated with cytosol. CEH activity and CEH protein were strongly correlated (r = 0.99) in different subcellular fractions. The small amount of CEH activity and protein associated with microsomes was further reduced by washing. Loss of more than 50% of microsomal activity by washing was also reported by Nilsson (1976), supporting the view that CEH activity associated with microsomes is primarily adsorbed soluble protein. This could also explain large variations in microsomal CEH activity reported by Neelon and Lack (1977). Low levels of CEH activity (and comparable protein) were also detected in the
FIG. 3. Cross-reactivity of cytosolic protein from various organs with polyclonal antibodies to liver CEH. An aliquot of 20 pg of ammonium sulfate precipitated protein was separated by SDSPAGE and probed with anti-liver CEH antiserum by Western blot analysis, as described in the Materials and methods. Lanes 1-8 represent kidney, testis, liver, pancreas, intestine, heart, serum, and adrenal, respectively. While the reaction was instantaneous in liver, the time required for the band to appear was 2-3 min for testis, kidney, and adrenal; 5-6 min for serum and heart; and 10-15 min for pancreas and intestine.
crude mitochondrial fraction (Table I), consistent with earlier reports of mitochondria1 CEH activity by Deykin and Goodman (1962). These results provide strong support for cytosolic localization of the predominant CEH activity of the liver. Tissue cross-reactivity Inasmuch as the primary focus of this report was immunological characterization of hepatic neutral CEH, we examined cross-reactivity of anti-liver CEH antiserum with cytosolic fractions from kidney, testis, liver, pancreas, intestine, heart, serum and adrenal. Ammonium sulfate precipitates of cytosol from these tissues were probed for cross-reactivity by Western blot analysis. As seen in Fig. 3, anti-liver CEH reacts with a 66-kDa protein from all the tissue samples used, except serum, indicative of the presence of proteins with similar molecular masses and some common epitopes. Serum contained two reactive bands, neither of which corresponded to 66 kDa. Absence of the 66-kDa band from serum rules out the possibility that it might be present in any of the other tissues as a result of contamination by serum. Weak and relatively slow reactions with pancreatic and intestinal proteins indicate a relatively low concentration in these tissues of antigenic epitopes shared with liver CEH, despite relatively high levels of CEH in these tissues. This view is supported by our previous report that antibodies to pancreatic CEH exhibited no crossreactivity with liver CEH purified by us (Ghosh and Grogan 1991). These results provide further evidence that the predominant CEH isoforms of liver and pancreas are different enzymes. Further comparison of hepatic CEH with isoforms in other tissues awaits the availability of complete protein (nucleotide) sequence for hepatic CEH. Preliminary results from work in progress suggest major differences in the C-terminal sequences of the hepatic and pancreatic enzyme.
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NOTES
Behrman, H.R., and Greep, R.O. 1972. Hormonal dependence of cholesterol ester hydrolase in the corpus luteum and adrenals. Horm. Metab. Res. 4: 206-209. Calame, K.B., Gallo, L., Cheriathundam, E., Vahouny, G.V., and Treadwell, C.R. 1975. Purification and properties of subunits of sterol ester hydrolase from rat pancreas. Arch. Biochem. Biophys. 168: 57-65. Carter, R.J., and Boyd, N.D. 1979. A comparison of methods for obtaining high yields of pure immunoglobulin from severely haemolysed plasma. J. Immunol. Methods, 26: 213-222. Cook, K.G., Colbran, R.J., Snee, J., and Yeaman, S.J. 1983. Cytosolic cholesterol ester hydrolase from bovine corpus luteum: its purification, identification and relationship to hormone sensitive lipase. Biochim. Biophys. Acta, 752: 46-53. Deykin, D., and Goodman, D.S. 1962. The hydrolysis of long chain fatty acid esters of cholesterol with rat liver enzymes. J. Biol. Chem. 237: 3649-3656. Durham, L.A., and Grogan, W.M. 1982. Temperature sensitivity of cholesteryl ester hydrolases EC-3.1.1.13 in the rat testis. Lipids, 17: 970-975. Durham, L.A., and Grogan, W.M. 1984. Chracterization of multiple forms of cholesteryl ester hydrolases EC-3.1 .l. 13 in the rat testis. J. Biol. Chem. 259: 7433-7438. Eto, Y., and Suzuki, K. 1972. Cholesterol esters in developing rat brain: enzymes in cholesterol ester metabolism. J. Neurochem. 19: 109-115.
803
Ghosh, S., and Grogan, W.M. 1989. Activation of rat liver cholesterol ester hydrolase by CAMP dependent protein kinase and protein kinase C. Lipids, 24: 733-736. Ghosh, S., and Grogan, W.M. sin1991. Rapid three step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme. Lipids, 26: 793-798. Martinez, M.J., and Botham, K.M. 1990. Cholesteryl ester hydrolase: three activities in the lactating rat mammary gland. Biochem. Soc. Trans. 18: 619-620. Neelon, N.J., and Lack, L. 1977. The effect of bile salts on the formation and hydrolysis of cholesterol esters by rat liver enzymes. Biochim. Biophys. Acta, 487: 137-144. Nilsson, A. 1976. Hydrolysis of chyle cholesterol esters with cell free preparations of rat liver. Biochim. Biophys. Acta, 450: 379-389. Pittman, R.C., and Steinberg, D. 1977. Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship. Biochim. Biophys. Acta, 487: 431-444. Schimke, R.T. 1975. Methods for analysis of enzyme synthesis and degradation in animal tissues. Methods Enzymol. 40: 241-266. Trzeciak, W.H., and Boyd, G.S. 1974. Activation of cholesteryl esterase in bovine adrenal cortex. Eur. J. Biochem. 46: 201-207. Voller, A., Bidwell, D.E., and Bartlett, A. 1979. In ELISA-a guide with abstracts of microplate applications. Dynatech Laboratories Inc., Alexandria, Va., U.S.A.
Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage Department of Clinical Biochemistry, University of Toronto and Toronto General Hospital, Toronto, Ont., Canada M5G 2C4 Received April 30, 1992 WU, T.-W., Wu, J., CAREY,D., and ZENG,L.-H. 1992. Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage. Biochem. Cell Biol. 70: 803-809. Rat ventricular myocytes have been isolated and cultured by two separate procedures. Using phase-contrast and electron microscopies, we illustrate that (a) definitive cell damage is produced when myocytes are exposed to xanthine oxidase - hypoxanthine and (b) purpurogallin between 0.25 and 1.0 mM prolongs survival of both myocyte preparations in a dose-dependent manner. The cytoprotection produced by 1 mM purpurogallin exceeds that given by 2 mM each of ascorbate, Trolox, and mannitol, or 24 200 IU superoxide dismutase/L and (or) 92 000 IU catalase/L. Furthermore, we noted, for the first time, that purpurogallin markedly protects rat aortic endothelial cells, a key target of free radical generation and attack. In contrast, Trolox has a negligible effect here. Mechanistically, we showed that purpurogallin inhibits urate formation by xanthine oxidase more potently than allopurinol. Also, the compound diminishes formation of superoxide-reduced cytochrome c. Therefore, purpurogallin is a potent protector of ventricular myocytes and aortic endothelial cells, both of which are important cells in the cardiovascular system. Key words: purpurogallin, endothelial cells, myocytes. WU, T.-W., WU, J., CAREY,D., et ZENG,L.-H. 1992. Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage. Biochem. Cell Biol. 70 : 803-809. Les myocytes ventriculaires de rat sont isolts et cultivts avec deux techniques distinctes. Utilisant la microscopie a contraste de phase et la microscopie tlectronique, nous illustrons (a) qu'un dommage cellulaire dtfinitif est produit XO, xanthine oxidase; TX, Trolox (Trolox ABBREVIATIONS: PPG, purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one); C; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyic acid); HBSS, Hanks' buffered saline solution; MEM, minimum essential medium; KRB, Krebs-Ringer bicarbonate buffer; PBS, sodium phosphate buffered saline (pH 7.4); TEM, transmission electron microscopy. ' ~ u t h o to r whom all correspondence should be sent at the following address: ES 3-404B, Toronto General Hospital, 200 Elizabeth St., Toronto, Ont., Canada M5G 2C4. Printed in Canada / Imprime au Canada
Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol SHOBHA GHOSHAND W. MCLEANGROGAN'
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Department of Biochemistry and Molecular Biophysics, Box 614, MCV Station, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0614, U.S.A. Received March 17, 1992
GHOSH, S., and GROGAN, W.M. 1992. Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol. Biochem. Cell Biol. 70: 800-803. Rabbit polyclonal antibodies were raised against rat liver bile salt-independent neutral cholesteryl ester hydrolase (CEH) and used for subcellular localization and immunological comparison with isoforms from other tissues. Antibodies exhibited a high degree of specificity for the liver CEH through all stages of purification and neutralized 70-80% of the activity of liver cytosolic CEH. They exhibited various levels of cross-reactivity with cytosolic proteins from other tissues, but reacted weakly with pancreatic and intestinal proteins and did not inhibit pancreatic CEH. Cytosol contained 78% of total cellular CEH activity and 75% of CEH immunoreactive protein. Washed microsomes contained 3% of CEH activity and 5% of CEH protein. Key words: cholesteryl esterase, polyclonal antibodies, rat liver, subcellular distribution.
GHOSH, S., et GROGAN, W.M. 1992. Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol. Biochem. Cell Biol. 70 : 800-803. Des anticorps polyclonaux de lapin sont ClevCs contre la cholesteryl ester hydrolase (CEH) neutre independante des sels biliaires de foie de rat et utilises pour la localisation subcellulaireet la comparaison immunologique avec les isoformes des autres tissus. Les anticorps manifestent un haut degre de specificit6 pour la CEH hepatique a tous les stades de la purification et ils neutralisent 70-80% de l'activite de la CEH cytosolique hkpatique. 11s exhibent divers taux de rkactivite avec les prottines cytosoliques d'autres tissus, mais ils reagissent faiblement avec les protkines pancrkatiques et intestinales et ils n'inhibent pas la CEH pancreatique. Le cytosol contient 78% de l'activite cellulaire totale de la CEH et 75% de la protkine CEH immunorCactive. Les microsomes renferment 3% de I'activitC CEH et 5% de la protiine CEH. Mots clks : cholesteryl esttrase, anticorps polyclonaux, foie de rat, distribution subcellulaire. [Traduit par la redaction]
Introduction Neutral cholesteryl ester hydrolases occur in many tissues, including liver (Deykin and Goodman 1962), pancreas (Calame et al. 1975), adrenals (Trzeciak and Boyd 1974), gonads (Durham and Grogan 1982; Behrman and Greep 1972), mammary glands (Martinez and Botham 1990), brain (Eto and Suzuki 1972), and corpus luteum (Cook et al. 1983), where they catalyze release of free cholesterol from cholesteryl ester, an intracellular storage form of cholesterol. Hepatic CEH, along with HMG-CoA reductase, cholesterol 7a-hydroxylase, and acyl-CoA:cholesterol acyltransferase, is a potentially important enzyme in regulation of the bile acid precursor (free cholesterol) pool in the liver. Multiple forms of CEH have been reported in testis (Durham and Grogan 1984), adrenals (Pittman and Steinberg 1977), and brain (Eto and Suzuki 1972), as well as liver (Deykin and Goodman 1962; Nilsson 1976). We have previously reported ABBREVIATIONS: CEH, cholesteryl ester hydrolase: HMG-CoA, TEMED, hydroxymethyl glutaryl coenzyme A; PI, isoeiectric N,N,N1, Nf-tetramethylethylenediamine; IgG, immunoglobulin G; PVDF, polyvinylidene difluoride; PEG, polyethylene glycol 8000; ABTS, 2,2'-azino-bis(3-ethylbenzthiazone-6-sulfonic acid); SDS, sodium dodecyl sulfate; kDa, kilodalton(s); CFA, complete Freund's adjuvant; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PBS, 100 mM phosphate buffer (pH 7.5) containing 150 mM NaCl. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
purification and characterization of a protein kinase activated CEH from rat liver cytosol, which did not require bile salt for activity (Ghosh and Grogan 1991). This CEH differed from pancreatic CEH with respect to bile salt dependence, subunit behavior, and PI, and did not crossreact with antibodies raised against rat pancreatic CEH (Ghosh and Grogan 1991). Although neutral bile saltindependent CEH activity has been reported in both cytosolic and particulate fractions from rat liver (Deykin and Goodman 1962; Nilsson 1976), the relationship between these activities has not been clarified. To characterize neutral hepatic CEH immunologically and determine its subcellular distribution, we have produced polyclonal antibodies to the purified enzyme. Using these antibodies we demonstrate that this enzyme is primarily cytosolic and accounts for at least 78% of total CEH activity in rat liver. Immunological comparison between enzymes from different organs is also described. Materials and methods Cholesteryl [l-'4~]oleate(56.6 mCi/mmol) was purchased from New England Nuclear (Boston, Mass.). All solvents were purchased from Fischer Scientific (Columbia, Md.). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, goat anti-rabbit IgG horse radish peroxidase conjugate, diaminobenzidine, and dye binding reagent for protein estimation were obtained from Bio-Rad Laboratories (Richmond, Calif.). Immobilon PVDF transfer mem-
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FIG. 1. Western blot analysis of rat liver neutral cytosolic CEH at different stages of purification. Proteins were separated by SDSPAGE and Western blots were developed as described in the Materials and methods. (A) Twenty-five micrograms of cytosolic protein; (B) 25 pg protein from 0 to 40% ammonium sulfate precipitate; (C) pooled and concentrated fractions from Mono S column, 12 pg protein; (D) concentrated fraction from gel permeation column, 2.7 pg protein (pure CEH).
branes were from Millipore (Bedford, Mass.). PEG, ammonium sulfate, ABTS, and Trizma base were purchased from Sigma Chemical Co. (St. Louis, Mo.). All other chemicals used were of analytical grade. Production of polyclonal antibodies CEH was purified from rat liver cytosol as described earlier (Ghosh and Grogan 1991). The pooled and concentrated fraction from the last step of purification (approximately 50-60 pg protein) was subjected to 5-20% SDS-polyacrylamide gel electrophoresis and the region corresponding to 66 kDa was cut and used for immunization. For the primary immunization, gel pieces were homogenized in saline and CFA (gel-saline-CFA, 3:1:4) and injected intradermally at 20-25 sites on the back of New Zealand rabbit on day 0. Subsequent booster immunizations were performed on day 14 and day 28 using the same amount of protein in gel pieces homogenized in incomplete adjuvant. The rabbit was test bled 7 days after the second booster and serum tested for antibodies using Western analysis as described below. A booster was given in incomplete adjuvant 7 days prior to subsequent bleedings. IgG was prepared from the test serum and preimmune serum (collected 7 days prior to immunization) by PEG precipitation as described by Carter and Boyd (1979). Subcellular fractionation Subcellular fractions were prepared from rat liver homogenate by differential centrifugation, as described earlier (Ghosh and Grogan 1989). All particulate fractions were washed once with the homogenizing buffer and resuspended in the same buffer for CEH assay and Western blot analysis. Western blot analysis Proteins separated by SDS-PAGE were electroblotted onto Immobilon-PVDF membrane using Integrated Separation System's semi-dry electroblotter. Western blots were developed following the standard protocol, using 5% nonfat dry milk in TBS (20 mM Tris-HC1 (pH 7.5) containing 150 mM NaCl) as the blocking solution, 1:200 dilution of immune serum in 1Yo gelatin in TBS as primary antibody, goat anti-rabbit IgG horse radish peroxidase conjugate as secondary antibody, and diaminobenzidine .as the color reagent. Western blots were scanned at 350 nm using a Shimadzu densitometer.
pg IgG Added FIG. 2. Neutralization of CEH activity in cytosol with anti-rat liver CEH IgG. Cytosolic protein (500-600 pg) was preincubated or immune ( a , anti-liver CEH) with 10-160 pg of preimmune (0) IgG and CEH activity was measured as described in Materials and methods. Data is plotted as percent CEH activity remaining, where 100% control was 2.3 + 0.1 nmol/h. Values are mean SEM of duplicates. Experiment was repeated with similar results.
*
ELISA Increasing concentrations of purified CEH protein were used to coat the plates and ELISA was carried out as described by Voller et al. (1979). ABTS was used as the color reagent. Neutralization assay Cytosolic protein (500-600 pg) was incubated with increasing concentrations of either preimmune or immune IgG (in PBS) at room temperature with vigorous shaking for 30 min. Following incubation, the volume was brought to 500 pL with 20 mM TrisHCl buffer (pH 7.5) containing 5 mM 2-mercaptoethanol and CEH activity was measured as described earlier (Ghosh and Grogan 1989). Protein determination Protein was measured by the Bio-Rad dye binding assay according to the manufacturer's instructions.
Results and discussion Specificity of antiserum Antiserum to liver CEH was tested for antibodies directed against neutral CEH from rat liver, by Western blot analysis. A positive reaction was observed with purified CEH from rat liver (Fig. ID). No reaction was observed with bovine serum albumin as a nonspecific control (data not shown). Antiserum also reacted with only one major protein band in cytosol and fractions representing different stages of purification of CEH (Fig. 1 , A-C). This band corresponded to a molecular mass of 66 kDa, the molecular mass of CEH (Ghosh and Grogan 1991). One or more minor bands were sometimes observed in cytosol and ammonium sulfate precipitate, the number and intensity of which increased upon storage, suggesting proteolytic degradation. These results confirm specificity of the polyclonal antibodies raised against CEH and suggest that this enzyme is immunologically different from other proteins in rat liver cytosol.
BIOCHEM. CELL BIOL. VOL. 70, 1992
802
TABLE 1. Subcellular distribution of liver neutral CEH
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-
Fraction
% total activity
% total CEH protein
Cytosol Unwashed microsomes Washed microsomes Crude mitochondria1 fraction
7k2.1
11k0.8
k D a 1 2 3 4
5 6 7 8
CEH activity and immunoreactive protein were measured in cytosolic and particulate fractions obtained from rat liver by differential centrifugation, as described in the Materials and methods. Data are presented as percent total liver activity, which was 391 nmol/h. CEH protein was measured by Western blot analysis, followed by densitomeric scanning. Values represent mean + SEM of two experiments in duplicate.
Specificity was also confirmed using ELISA. A positive linear correlation was observed between protein concentration of pure CEH and absorbance at 405 nm (r = 0.93) in a standard ELISA (data not shown). Neutralization of enzyme activity To confirm that antibodies were directed against CEH and to determine the fraction of activity represented by the CEH which we have purified, the capacity of antibodies to inhibit cytosolic CEH activity was examined. Whereas serum alone was found to inhibit CEH (S. Ghosh and W.M. Grogan, unpublished results), purified immune and preimmune control IgG were used in this study. Incubation with increasing concentrations of immune IgG resulted in increasing inhibition of CEH activity relative to preimmune control IgG (Fig. 2). Inhibition by 70-80% was observed at the highest IgG concentration used, strongly suggesting that the enzyme purified by us is the major cytosolic CEH in rat liver. In a separate experiment, pancreatic CEH was not significantly inhibited (81 k 5070, activity remaining, versus 37 10070,activity remaining in inhibited liver cytosol) by a single concentration of anti-liver CEH IgG (100 pg). These results support conclusions in our recent report (Ghosh and Grogan 1991) in which, based on high yield and specific activity, we suggested that this CEH is the predominant activity in rat liver cytosol. Some inhibition was also observed with preimmune IgG (Fig. 2). This is similar to the inhibition of other enzymes by immunoglobulins reported by others and attributed to nonspecific interaction of immunoglobulins with unstable enzymes (Schimke 1975). Subcellular distribution of CEH in rat liver There has been considerable confusion and some controversy concerning the relative contributions of cytosolic and particulate forms of CEH in the liver. We have addressed this question directly by measuring CEH activity and enzyme mass in liver cytosol, the crude mitochondrial fraction, and microsomes. As seen in Table 1,78% of total CEH activity and 75% of CEH protein were associated with cytosol. CEH activity and CEH protein were strongly correlated (r = 0.99) in different subcellular fractions. The small amount of CEH activity and protein associated with microsomes was further reduced by washing. Loss of more than 50% of microsomal activity by washing was also reported by Nilsson (1976), supporting the view that CEH activity associated with microsomes is primarily adsorbed soluble protein. This could also explain large variations in microsomal CEH activity reported by Neelon and Lack (1977). Low levels of CEH activity (and comparable protein) were also detected in the
FIG. 3. Cross-reactivity of cytosolic protein from various organs with polyclonal antibodies to liver CEH. An aliquot of 20 pg of ammonium sulfate precipitated protein was separated by SDSPAGE and probed with anti-liver CEH antiserum by Western blot analysis, as described in the Materials and methods. Lanes 1-8 represent kidney, testis, liver, pancreas, intestine, heart, serum, and adrenal, respectively. While the reaction was instantaneous in liver, the time required for the band to appear was 2-3 min for testis, kidney, and adrenal; 5-6 min for serum and heart; and 10-15 min for pancreas and intestine.
crude mitochondrial fraction (Table I), consistent with earlier reports of mitochondria1 CEH activity by Deykin and Goodman (1962). These results provide strong support for cytosolic localization of the predominant CEH activity of the liver. Tissue cross-reactivity Inasmuch as the primary focus of this report was immunological characterization of hepatic neutral CEH, we examined cross-reactivity of anti-liver CEH antiserum with cytosolic fractions from kidney, testis, liver, pancreas, intestine, heart, serum and adrenal. Ammonium sulfate precipitates of cytosol from these tissues were probed for cross-reactivity by Western blot analysis. As seen in Fig. 3, anti-liver CEH reacts with a 66-kDa protein from all the tissue samples used, except serum, indicative of the presence of proteins with similar molecular masses and some common epitopes. Serum contained two reactive bands, neither of which corresponded to 66 kDa. Absence of the 66-kDa band from serum rules out the possibility that it might be present in any of the other tissues as a result of contamination by serum. Weak and relatively slow reactions with pancreatic and intestinal proteins indicate a relatively low concentration in these tissues of antigenic epitopes shared with liver CEH, despite relatively high levels of CEH in these tissues. This view is supported by our previous report that antibodies to pancreatic CEH exhibited no crossreactivity with liver CEH purified by us (Ghosh and Grogan 1991). These results provide further evidence that the predominant CEH isoforms of liver and pancreas are different enzymes. Further comparison of hepatic CEH with isoforms in other tissues awaits the availability of complete protein (nucleotide) sequence for hepatic CEH. Preliminary results from work in progress suggest major differences in the C-terminal sequences of the hepatic and pancreatic enzyme.
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NOTES
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Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage Department of Clinical Biochemistry, University of Toronto and Toronto General Hospital, Toronto, Ont., Canada M5G 2C4 Received April 30, 1992 WU, T.-W., Wu, J., CAREY,D., and ZENG,L.-H. 1992. Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage. Biochem. Cell Biol. 70: 803-809. Rat ventricular myocytes have been isolated and cultured by two separate procedures. Using phase-contrast and electron microscopies, we illustrate that (a) definitive cell damage is produced when myocytes are exposed to xanthine oxidase - hypoxanthine and (b) purpurogallin between 0.25 and 1.0 mM prolongs survival of both myocyte preparations in a dose-dependent manner. The cytoprotection produced by 1 mM purpurogallin exceeds that given by 2 mM each of ascorbate, Trolox, and mannitol, or 24 200 IU superoxide dismutase/L and (or) 92 000 IU catalase/L. Furthermore, we noted, for the first time, that purpurogallin markedly protects rat aortic endothelial cells, a key target of free radical generation and attack. In contrast, Trolox has a negligible effect here. Mechanistically, we showed that purpurogallin inhibits urate formation by xanthine oxidase more potently than allopurinol. Also, the compound diminishes formation of superoxide-reduced cytochrome c. Therefore, purpurogallin is a potent protector of ventricular myocytes and aortic endothelial cells, both of which are important cells in the cardiovascular system. Key words: purpurogallin, endothelial cells, myocytes. WU, T.-W., WU, J., CAREY,D., et ZENG,L.-H. 1992. Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage. Biochem. Cell Biol. 70 : 803-809. Les myocytes ventriculaires de rat sont isolts et cultivts avec deux techniques distinctes. Utilisant la microscopie a contraste de phase et la microscopie tlectronique, nous illustrons (a) qu'un dommage cellulaire dtfinitif est produit XO, xanthine oxidase; TX, Trolox (Trolox ABBREVIATIONS: PPG, purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one); C; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyic acid); HBSS, Hanks' buffered saline solution; MEM, minimum essential medium; KRB, Krebs-Ringer bicarbonate buffer; PBS, sodium phosphate buffered saline (pH 7.4); TEM, transmission electron microscopy. ' ~ u t h o to r whom all correspondence should be sent at the following address: ES 3-404B, Toronto General Hospital, 200 Elizabeth St., Toronto, Ont., Canada M5G 2C4. Printed in Canada / Imprime au Canada
