Chem. Res. Toxicol. 1992, 5, 406-410
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(31) Sheldon, R. A., and Kochi, J. K. (1981) Metal-catalyzed oxidations of organic compounds, pp 35-38, Academic Press, New York. (32) Winston, G. W., and Cederbaum, A. I. (1983) NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. J. Biol. Chem. 258, 1514-1519. (33) Ravindranath, V., Anandatheerthavarada, H. K., and Shankar, S. K. (1990) NADPH cytochrome P450 reductase in rat, mouse, and human brain. Biochem. Pharmacol. 39, 1013-1018. (34) Kennedy, C. H., and Mason, R. P. (1990) A reexamination of the cytochrome P-450-catalyzed free radical production from a
dihydropyridine. J . Biol. Chem. 265, 11425-11428. (35) Fukuto, J. M., Kumagai, Y., and Cho, A. K. (1991) Determination of the mechanism of demethylenation of (methylenedioxy)phenyl compounds by cytochrome P450 using deuterium isotope effects. J . Med. Chem. 34, 2871-2876. (36) Steele, T. D., Brewster, W. K., Johnson, M. P., Nichols, D. E., and Yim, G. K. W. (1991) Assessment of the role of a-methylepinine in the neurotoxicity of MDMA. Pharmacol. Biochem. Behau. 38, 345-351. (37) Hicks, M., and Gebicki, J. M. (1986) Rate constants for reaction of hydroxyl radicals with Tris, Tricine and Hepes buffers. FEBS Lett. 199, 92-94.
The Calcium-Binding Protein Calreticulin I s Covalently Modified in Rat Liver by a Reactive Metabolite of the Inhalation Anesthetic Halothane Lynn E. Butler,? David Thomassen,' Jackie L. Martin,tJ Brian M. Martin,§ J. Gerald Kenna,t*"and Lance R. Pohl*lt Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, and Clinical Neurosciences Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, and Department of Anesthesiology and Critical Care Medicine, T h e Johns Hopkins Medical Institutions, Baltimore, Maryland 21205 Received December 23, 1991
A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.17% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.
Introductlon It has been estimated that more than 600 drugs cause hepatic injury (1,2). These compounds can be classified as being either intrinsic or idiosyncratic hepatotoxins (3). In both cases, however, reactive metabolites have been implicated in producing the toxicity (4). For example, reactive metabolites of intrinsic hepatotoxic agents are thought to cause toxicity by covalently altering cellular macromolecules either directly or indirectly. It is believed that they indirectly cause these modifications by causing lipid peroxidation and protein cross-linking and Sthiolation (4-6). These reactions may lead to the inactivation of enzymes, to the disruption of intracellular cal*Address correspondence to this author at the Laboratory of Chemical Pharmacology, NHLBI, NIH, Building 10, Room 8N 115, Bethesda, MD 20892. 'Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute. t Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions. 9 Clinical Neurosciences Branch, National Institute of Mental Health. 11 Present address: Department of Pharmacology and Toxicology, St. Mary's Hospital Medical School, Norfolk Place, London W2 IPG.
cium homeostasis, or to a general loss of cellular membrane integrity (4-7). Idiosyncratic drug-induced hepatic damage also might be attributed to similar processes that for some reason are very host dependent. It could be due to the presence of abnormally high levels of enzymes that convert the drug into a reactive metabolite, a mutant form of the enzyme that is catalytically more active than the normal enzyme, or abnormally low levels or activities of enzymes that detoxify either the reactive metabolite or its precursor. Alternatively, a reactive metabolite may produce an idiosyncratic drug-induced hepatotoxicity by an allergic or hypersensitivity reaction that is directed against a covalently altered tissue macromolecule (neoantigen) (3). Lack of knowledge about the target macromolecules in the liver of reactive metabolites of drugs has slowed the elucidation of the mechanism of hepatotoxicity (4). In this regard, we have started to characterize liver neoantigens associated with the idiosyncratic hepatotoxicity produced by the inhalation anesthetic, halothane. Patients with this toxicity have been shown by immunoblotting to have serum antibodies that react with at least five distinct rat liver microsomal polypeptide fractions (100, 76, 59, 57, and 54 kDa), which are covalently altered by the trifluoroacetyl
This article not subject to U.S. Copyright. Published 1992 by the American Chemical Society
Covalent Modification of Calreticulin by Halothane (TFA)' halide metabolite of halothane (8). Since these antibodies are specific to patients with halothane hepatitis, it is thought that this toxicity may have an immunopathological basis (9, IO). Recently, the TFA-59-kDa neoantigen has been purified and identified as a carboxylesterase (II), which appears to correspond to isoforms E l (12) and ES-8/ES-10 (13). In the present study, we report a general procedure for the isolation of TFA liver microsomal proteins. Using this approach, we describe the purification and characterization of a 63-kDa protein target of halothane and have identified it as calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum.
Experimental Procedures Materials. Sera were obtained from patients with a clinical diagnosis of halothane hepatitis and from control patients, as described in detail elsewhere (14). Hapten-specific anti-TFA sera was produced by immunizing rabbits with TFA-rabbit serum albumin as previously described (15). Purification of the 63-kDa Protein. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) (44 animals, 210-230 g) were treated with halothane (Halocarbon Laboratories, Hackensack, NJ, purified by distillation) (20 mmol/kg, ip, as a 21.5% solution in sesame oil). After 16-18 h, livers were removed (370 g) and homogenized in 740 mL of 100 mM Tris-acetate, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA (buffer A). The homogenate was centrifuged a t lOOOOg a t 4 "C for 35 min, and the resulting supernatant was centrifuged at lOOOOOg for 120 min. After the pellet was washed, it was resuspended in 370 mL of 10 mM Tris-HC1, pH 7.5, containing 20% glycerol and 0.1 mM EDTA and stored a t -80 "C until used. A 115-mL aliquot of the microsomes (3 g of protein) was diluted with 3 volumes of phosphate-buffered saline. The mixture was centrifuged at lOO00Og a t 4 "C for 120 min. The resulting pellet was resuspended in 420 mL of 10 mM Tris-HC1, pH 7.5, containing 0.1 mM EDTA and 0.1% (w/v) DOC (buffer B) and gently stirred at 4 "C for 1 h. After centrifugation at l00OOOg at 4 "C for 2 h, the supernatant (DOC extract) (420 mL, 1.4 mg protein/mL) was applied at a flow rate of 3 mL/min to a DEAE-Sepharose (Piscataway, NJ) column (5 X 23 cm) that had been equilibrated with buffer B. The column was washed with 1 column volume of buffer B and 1 column volume of 10 mM Tris-HC1, pH 7.5, containing 0.1 mM EDTA (buffer C). Proteins were eluted (25 &/fraction) with a 4-L linear solvent gradient of 0 4 . 5 M sodium chloride in buffer C and were continuously monitored by their absorption at 275 nm. The fractions were analyzed by SDS/PAGE for their protein composition and by immunoblotting for their reactivity with hapten-specific anti-TFA antibodies. The TFA-63-kDa protein eluted from the column at 0.34-0.38 M NaCl and was contaminated with a 100-kDa protein. The mixture (350 mL) was concentrated to 35 mL in an Amicon ultrafiltration cell using a PM 30 membrane (Danvers, MA) and partially desalted by dilution with 150 mL of 20 mM sodium phosphate, pH 7.0, followed by ultrafiltration to a volume of 25 mL. The TFA-63-kDa protein was further purified by anion-exchange HPLC chromatography on a Bio-Gel TSK DEAE-5-PW column (Bio-Rad, Richmond, CA) (2.15 X 15 cm) that had been equilibrated with 20 mM sodium phosphate, pH 6.5 (buffer D), a t a flow rate of 5 mL/min. After injection of the sample, the column was washed with buffer D for 5 min, followed by a 45-min linear solvent program of 0 . 6 M sodium chloride in buffer D. Absorbance of the eluent was monitored at 280 nm. One-minute fractions were collected throughout the program and analyzed by SDS/PAGE. The TFA-63-kDa protein eluted from the column a t approximately 0.47 M NaCl. Since it was still partially contaminated with the 100-kDa protein, the HPLC purification step was repeated. The TFA-63-kDa protein was recovered in a final yield of 3.6 mg (0.1% of initial microsomal Abbreviations: TFA, trifluoroacetylated;DOC,sodium deoxycholate, SDS PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; ELILA, enzyme-linked immunosorbent assay; HACBP, high-affinity Caz+-bindingprotein; CRP55, 55-kDa calcium-binding reticuloplasmin; PTH, phenylthiohydantoin.
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 407 protein). The unlabeled 63-kDa protein was purified in a similar manner from liver microsomes (3 g of protein) of control rats in a final yield of 8.3 mg (0.3% of initial microsomal protein).
Amino Acid Sequence Analysis of the 63-kDa Protein. The amino acid sequences of the N-terminal and intemal peptides were obtained by automated Edman degradation with the use of an Applied Biosystems Model 470 A gas-phase sequencer (Foster City, CA) equipped with an online Model 120 A P T H amino acid analyzer. Normal program 03R P T H was employed as provided by Applied Biosystems. The protein was precipitated by the addition of 4 volumes of ice cold absolute ethanol. After centrifugation, the pellet was resuspended in 900 pL of water and was reprecipitated by the addition of 4 volumes of ice-cold ethanol. A portion of the sample was taken for N-terminal amino acid sequence analysis. The remainder of the sample was treated with trypsin (Sigma, St. Louis, MO, type XI, T-1005 from bovine pancreas) or Asp-N (Boehringer Mannheim, Indianapolis, IN) to produce internal peptides that could be sequenced. For hydrolysis with trypsin, 100 pg of protein was mixed with 200 pL of 1 M urea for 5 min, followed by the addition of 100 mM potassium phosphate, pH 8.0, trypsin (5 pg), and 20 mM methylamine in a final volume of 800 pL. After incubation for 18 h at 37 "C, peptides were isolated from the reaction mixture by HPLC on a Vydac (2-18 column (The Nest Group, Southborough, MA) (0.46 cm X 25 cm), with a 60-min linear solvent program of 9O:lO (v/v) solvent A (0.12% trifluoroacetic acid)/solvent B (0.10% trifluoroacetic acid in acetonitrile) to 2080 (v/v) solvent A/solvent B, at a flow rate of 1mL/min. For the hydrolysis with Asp-N, 122 pg of protein was mixed with 500 pL of 10 mM potassium phosphate, pH 6.8, containing 10% (v/v) acetonitrile. The mixture was heated at 100 "C for 5 min, followed by incubation with Asp-N (2.4 pg) for 18 h at 37 "C. Peptides were isolated by HPLC as described for the incubations with trypsin. Antiserum Preparation. Antisera were raised against the TFA-63-kDa protein by mixing 77 pg of the protein in anionexchange HPLC eluent with 3 volumes of Freund's complete adjuvant and injecting the mixture intradermally into multiple sites on the back of a 2.5-kg female New Zealand White rabbit (Dutchland, Denver, PA). After 7 weeks, sera were collected weekly for 6 weeks. cDNA Isolation and Sequencing. A X g t l l cDNA library prepared from the liver of a male Sprague Dawley rat (Clontech, Palo Alto,CA) was screened with anti-63-kDa rabbit serum, using blocking and washing conditions described for immunoblotting experiments (8). Selection of 63-kDa related clones were confirmed by their hybridization to biotinylated (Clontech Kit) synthetic oligonucleotides derived from internal peptides of the 63-kDa protein. Isolated EcoRI inserts were subcloned into the pGem 7f(+) vector (Promega, Madison, WI) and sequenced by the double-stranded method using SP6 and T7 primers (Promega) and the Sequenase Kit 2.0 (United States Biochemical, Cleveland, OH). Sequences were extended using primers synthesized to progressive distal regions of the sequenced oligonucleotide strand. Sequences were assembled using Microgenie Sequence Software (Beckman, Palo Alto, CA). Other Methods. The reaction of serum antibodies from control and halothane hepatitis patients with the purified native and TFA-63-kDa proteins was measured by a previously described ELISA method (14). SDS/PAGE and immunoblotting were performed as reported elsewhere, except that standard-length gels were used instead of minigels (8). Calcium binding to the 63-kDa protein was determined by autoradiography of 45Caoverlays of immunoblots by the procedure of Maruyama et al. (16). Protein was determined according to the method of Lowry et al. (17) with bovine serum albumin as a standard.
Results When liver microsomes from halothane-treated rats were treated with 0.1% DOC, TFA protein fractions of molecular masses of approximately 57 kDa, 59 kDa, 80 kDa [previously designated as the 76-kDa fraction (S)], and 100 kDa were extracted (Figure 1). A 63-kDa protein fraction also was extracted from the liver microsomes by this procedure. It appeared to be covalently modified by the trifluoroacetyl halide metabolite of halothane because it
408 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
Butler et al.
chromatographed on an anion-exchange HPLC column to purify the protein from the 100-kDa protein and other minor contaminants (Figure 3 and Figure 4, part 1). A similar approach was used to purify the unlabeled 63-kDa protein from livers of untreated rats (Figure 4). The native protein in contrast to the TFA-63-kDa protein did not react with the anti-TFA antibodies (Figure 4, part 3). An antibody raised against the TFA-63-kDa protein reacted strongly with the native 63-kDa protein, confirming that the proteins were highly related if not identical (Figure 4, part 2). Only 1of 40 patients diagnosed with halothane hepatitis had serum antibodies that reacted with the TFA-63-kDa protein at a level significantly higher than did serum antibodies from a variety of control patients (results not shown). At the time that this work was begun, the amino acid sequences of the N-terminal and of several internal peptides of the 63-kDa protein did not show high homology with any known proteins. In order to learn more about the structure of the protein, a 1.3-kb cDNA clone was isolated from a X g t l l rat liver library that was screened with anti-TFA 63-kDa sera and its nucleotide sequence was determined. The sequence encoded for an open reading frame of 248 amino acids, contained a potential asparagine glycosylation site, and ended with the sequence KDEL, which is a retention signal for soluble luminal proteins of the endoplasmic reticulum (Figure 5) (19). Searches of protein and DNA data bases with this sequence information revealed that the encoded sequence of the 1.3-kb clone was 98% idential to that of the recently published sequence of a murine cDNA encoding for the calciumbinding protein, calreticulin (Figure 5) (20). The finding that the amino acid sequences of the N-terminal and that of several internal peptides of the 63-kDa protein corresponded exactly to the encoded sequences of the two cDNAs confirmed that the 63 kDa was calreticulin (Figure 5). Moreover, autoradiography of W a overlays of immunoblots of the purified 63-kDa protein revealed that it was a calcium-binding protein (results not shown).
B
A
-146 100-
-100
80-
-80 -63 -59 - 57
357= 50-
-50
47-
1 2 3
1 2 3
Figure 1. Extraction of "FA proteins with 0.1%DOC from liver microsomes of halothane-treated rats. Liver microsomes before extraction (lanes 1) and after extraction (lanes 2), and the extract (lanes 3), were separated into constitutive polypeptides by SDS/PAGE and stained with Coomassie Blue (part A) or were transblotted to nitrocellulose and immunoreacted with anti-TFA antibodies (part B). Each lane contained 50 pg of protein.
reacted with anti-TF'A antibodies as did most of the other extracted proteins (Figure 1B). Major protein fractions in the range of 50 kDa were not appreciably solubilized by this procedure, probably because they were integral membrane proteins such as cytochromes P-450,in contrast to the extracted proteins, which were likely peripheral or luminal proteins (18). The extract of TFA proteins was separated into partially purified fractions by chromatography on a DEAE-Sepharose anion-exchange column (Figure 2). The fractions containing the TFA-63-kDa protein were combined and
Discussion In this study a procedure has been described that should be useful for the rapid isolation and purification of several 100 kDa
/
63 kDa
gl
80 kDa
v)
LA!-----
58 kDa
IN
9.10 100
40
50
60
70
80
90
I
I
I 60
I
70
I 80
I
40
I 50
I
30
90
100
30
Fraction Number
Figure 2. DEAE-Sepharose anion-exchange chromatography of the DOC extract of liver microsomes from halothane-treatedrats. The lower panel is the SDS/PAGE analysis of the eluting protein fractions, which were stained with Coomassie Blue.
Covalent Modification of Calreticulin by Halothane 1
yl.0
63 kDa
Chem. Res. Toxicol., Vol. 5, No. 3,1992 409 CR
-17 MLLSVPLLULLGLAAA
CR
DPAIYFKEQFLDGDAWTNESKHKSDFGKFVLSSGKFYGDLEKDKGW 1
50
2
43kDa %
100
CR
TSQDARFYALSAKFEPFSNKGQTLWQFTVKHEQNIDCGGGYVKLFPSGL
CR
WKDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDE 150
63kDA
THLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDAAKPE
CR
FTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDAAKPE 2 0 0
63kDA
DWDERAKIDDFTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQ
3 __..*=
J .___* 0
I
I
20
10
CR
I
30
40
50
Time, min
Figure 3. HPLC anion-exchangechromatography separation of the TFA-63-kDa protein from the TFA-100-kDa protein. The inset is a protein-stained SDS/PAGE gel of the mixture of TFA-63-kDa and TFA-100-kDa proteins isolated by DEAE-Sepharose anion-exchange chromatography (Figure 2), before they were separated by HPLC anion-exchange. The molecular mass standards are on the left and the mixture of TFA-63-kDa and TFA-100-kDa proteins are on the right side of the gel.
1
63 kDa -4lllIlb
2
3
-
97 kDa
-
66 kDa
-
43 kDa
H C H C H C Figure 4. Purified TFA-63-kDa and native 63-kDa proteins. Part 1is an SDS/PAGE gel stained for protein with Coomassie Blue; part 2 is a transblot of an SDS/PAGE gel immunoreacted with anti-TFA-63-kDa antibodies; and part 3 is a transblot of an SDS/PAGE gel immunoreacted with anti-TFA antibodies. Lane H contains the TFA-63-kDa protein purified from livers of halothane-treated rats, while lane C contains the native 63-kDa protein purified from livers of untreated rats.
of the liver microsomal protein targets of the reactive trifluoroacetylhalide metabolite of halothane. The method has been used to purify a native and TFA-63-kDa protein, which has been identified as calreticulin. This protein appears to be identical to calregulin (21), HACBP (22), CRP55 (20), and a 60-kDa Ro/SS-A protein autoantigen associated with systemic lupus erythematosus and Sjogren’s syndrome (23). A similar approach has been described briefly for the purifications of the 59-kDa (24) and 58-kDa (25)protein targets of halothane. At the present time, it is not clear what role the TFAcalreticulin has in halothane hepatitis, since only 1of 40 patients diagnosed with this toxicity had serum antibodies that reacted with it. The low level of reactivity of the patient’s sera with rat TFA-calreticulin is probably not due to species differences between rat and human forms of this protein, because they appear to be at least 90% homologous (23). Instead, TFA-calreticulin may be less immunogenic than the other TFA-proteins recognized by halothane hepatitis patients’ serum antibodies. One possible
DWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQ 2 50 -4
60
5-
63kDA
NPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDANIYAYDSFAVLGLD
CR
NPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDANIYAYDSFAVLGLD 300
63kDA
LWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRL
CR
LWQVKSGTIFDNFLITNDEAYAEEFGNETWGWKAAEKQMKDKQDEEQRL 3 50
63kDA
KEEEEDKKRKEEEEAEDKEDEDDRDEDEDEEDEKEEDEEDATGQAKDEL
CR
KEEEEDKKRKEEEEAEDKEDDDDRDEDEDEEDEKEEDEEESPGQAKDEL
+
6
**t
399
Figure 5. Comparison of the deduced amino acid sequence of the partial-length rat liver 1.3-kb cDNA of the 63-kDa protein with that of a full-length murine cDNA of calreticulin (CR) (20). The underlined residues correspond to sequences derived from Edman degradation of the 63-kDa protein: sequence 1was derived from the N-terminal of the 63-kDa protein; sequences 3,4, and 6 were derived from tryptic peptides of the 63-kDa protein; sequences 2 and 5 were derived from digestion of the 63-kDa protein with Asp-N. A potential glycosylation site is indicated by (+) a t residue 327. The (*) indicate conservative substitutions, while the (t)indicates a nonconservative substitution.
explanation for this is that TFA-calreticulin appears to have a lower level of TFA groups bound covalently to it than other TF’A-neoantigens (26). This might be explained by a report indicating that calreticulin is concentrated in a specific domain of the endoplasmic reticulin or calciosomes (27), which might be farther removed from the cytochromes P-450 that activate halothane to trifluoroacetyl halide than are the other target proteins of this metabolite. It remains to be determined whether TFA binding to calreticulin alters its physiological functions and contributes to the development of halothane hepatitis. Calreticulin appears to be an important calcium-binding protein in the endoplasmic reticulum of most nonmuscle cells (28) and in the sarcoplasmic reticulum of both smooth muscle (29) and skeletal muscle cells (20). Consequently, it may have a role in modulating intracellular activities attributed to calcium, such as cell signaling (30), protein folding (31), protein secretion (32), and cell killing by perturbing cytoskeletal organization, impairing mitochondrial function, and activating phospholipases, proteases, and endonucleases (6). In this regard,it has been reported that halothane exposure to guinea pigs caused a reduction in microsomal calcium sequestration and an increase in hepatic calcium content, which was proportionate to the severity of liver necrosis (33). In addition, calreticulin has been shown recently to bind to flavin-containing monooxygenase (=), the integrin a subunits (%), and a vitamin K-dependent glycoprotein in plasma (36) and may be involved in the regulation of the activities of these proteins. It is possible that calreticulin also may be a target of the reactive metabolites of other compounds, especially those that are known to alter intracellular calcium homeostasis (4-6). Moreover, calreticulin may have a role in drug-induced lupus (37), since a subset of patients with lupus erythematosus have been shown recently to have autoantibodies directed against calreticulin (23).
410 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
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Acknowledgment. D.T. was supported by research funds from Anaquest. We thank Dr. James R. Gillette for valuable suggestions in editing the manuscript.
(20) Smith, M. J., and Koch, G. L. (1989) Multiple zones in the
References
245-251. (22) Fliegel, L., Burns, K., MacLennan, D. H., Reithmeier, R. A., and Michalak, M. (1989) Molecular cloning of the high affinity calci-
(1) Stricker, B. H. Ch., and Spoelstra, P. (1985) Drug-induced hepatic injury. A comprehensive survey of literature on adverse drug reactions up to January 1985, Elsevier, Amsterdam. (2) Zimmerman, H. J. (1990) Update of hepatotoxicity due to classes
of drugs in common clinical use: non-steroidal drugs, anti-inflammatory drugs, antibiotics, antihypertensives, and cardiac and psychotropic agents. Semin. Liver Dis. 10, 322-338. (3) Zimmerman, H. J. (1978) Hepatotoxicity. The Adverse Effects of Drugs and other Chemicals on the Liver, pp 91-121, Appleton-Century-Croft, New York. (4) Nelson, S. D., and Pearson, P. G. (1990) Covalent and noncovalent interactiom in acute lethal cell injury caused by chemicals. Annu. Rev. Pharmacol. Toxicol. 30, 169-195. (5) Reed, D. J. (1990) Glutathione: Toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30, 603-631. (6) Nicotera, P., Bellomo, G., and Orrenius, S. (1990) The role of Ca2+in cell killing. Chem. Res. Toxicol. 3, 484-494. (7) Tirmenstein, M. A., and Nelson, S. D. (1990) Acetaminopheninduced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides. J . Biol. Chem. 265, 3059-3065. (8) Kenna, J. G., Satoh, H., Christ, D. D., and Pohl, L. R. (1988) Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane. J . Pharmacol. Exp. Ther. 245, 1103-1109. (9) Pohl, L. R., Satoh, H., Christ, D. D., and Kenna, J. G. (1988) The immunologic and metabolic basis of drug hypersensitivities. Annu. Rev. Pharmacol. Toxicol. 28, 367-87. (10) Pohl, L. R. (1990)Drug-induced allergic hepatitis. Semin. Liver Dis. 10, 305-315. (11) Satoh, H., Martin, B. M., Schulick, A. H., Christ, D. D., Kenna, J. G., and Pohl, L. R. (1989) Human anti-endoplasmic reticulum antibodies in sera of halothane hepatitis patients are directed against a trifluoroacetylated carboxylesterase. Proc. Natl. Acad. Sci. U.S.A.86, 322-326. (12) Harano, T., Miyata, T., Lee, S., Aoyagi, H., and Omura, T. (1988) Biosynthesis and localization of rat liver microsomal carboxyesterase El. J . Biochem. 103, 149-155. (13) Mentlein, R., Ronai, A., Robbi, M., Heymann, E., and von Deimling, 0. (1987) Genetic identification of rat liver carboxylesterases isolated in different laboratories. Biochim. Biophys. Acta 913, 27-38. (14) Martin, J. L, Kenna, J. G., and Pohl, L. R. (1990) Antibody assays for the detection of patients sensitized to halothane. Anesth. Analg. 70, 154-159. (15) Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., and Pohl, L. R. (1985) Immunological studies on the mechanism of halothane-induced hepatotoxicity: Immunohistochemical evidence of trifluoroacetylated hepatocytes. J . Pharmacol. Exp. Ther. 233,857-862. (16) Maruyama, K., Mikawa, T., and Ebashi, S. (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J . Biochem. (Tokyo)95, 511-519. (17) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J . B i d . Chem. 193, 265-275. (18) Weihing, R. R., Manganiello, V. C., Chiu, R., and Phillips, A. H. (1972) Purification of hepatic microsomal membranes. Biochemistry 11, 3128-3135. (19) Pelham, H. R. (1990) The retention signal for soluble proteins of the endoplasmic reticulum. Trends.Biochem. Sci. 15,483-486.
sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO J . 8, 3581-3586. (21) Khanna, N. C., Tokuda, M., and Waisman, D. M. (1987) Comparison of calregulins from vertebrate livers. Biochem. J . 242,
um-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J . B i d . Chem. 264, 21522-21528. (23) McCauliffe, D. P., Zappi, E., Lieu, T. S., Michalak, M., Sontheimer, R. D., and Capra, J. D. (1990) A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia "memory molecule". J . Clin. Invest. 86, 332-335. (24) Medda, S., Chemelli, R. M., Martin, J. L., Pohl, L. R., and Swank, R. T. (1989) Involvement of the carboxyl-terminal propeptide of @-glucuronidasein ita compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach. J . Biol. Chem. 264, 15824-15828. (25) Martin, J. L., Pumford, N. R., LaRosa, A. C., Martin, B. M., Gonzaga, H. M., Beaven, M. A., and Pohl, L. R. (1991) A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-a but has no activity. Biochem. Biophys. Res. Commun. 178, 679-685. (26) Pohl, L. R., Thomassen, D., Pumford, N. R., Butler, L. E., Satoh, H., Ferrans, V. J., Perrone, A., Martin, B. M., and Martin, J. L. (1991) Hapten carrier conjugates associated with halothane hepatitis. Adu. Exp. Biol. Med. 283, 111-120. (27) Treves, S., De Mattei, M., Landfredi, M., Villa, A,, Green, N. M., MacLennan, D. H., Meldolesi, J., and Pozzan, T. (1990) Calreticulin is a candidate for a calsequestrin-like function in Ca2+-storage compartments (calciosomes) of liver and brain. Biochem. J . 271,473-480. (28) Khanna, N. C., and Waisman, D. M. (1986) Development of a radioimmunoassay for quantitation of calregulin in bovine tissues. Biochemistry 25, 1078-1082. (29) Milner, R. E., Baksh, S., Shemanko, C., Carpenter, M. R., Smillie, L., Vance, J. E., Opas, M., and Michalak, M. (1991) Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J . B i d . Chem. 266, 7155-7165. (30) Berridge, M. J. (1989) The Albert Lasker Medical Awards. Inositol trisphosphate, calcium, lithium, and cell signaling. JAMA, J . Am. Med. Assoc. 262, 1834-1841. (31) Lodish, H. F., and Kong, N. (1990) Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum. J . Biol. Chem. 265, 10893-10899. (32) Booth, C., and Koch, G. (1989) Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59, 729-737. (33) Farrell, G. C., Mahoney, J., Bilous, M., and Frost, L. (1988) Altered hepatic calcium homeostasis in guinea pigs with halothane-induced hepatotoxicity. J . Pharmacol. Exp. Ther. 247, 751-756. (34) Guan, S., Falick, A. M., Williams, D. E., and Cashman, J. R. (1991) Evidence for complex formation between rabbit lung flavin-containing monooxygenaseand calreticulin. Biochemistry 30, 9892-9900. (35) Rojiani, M. V., Finlay, B. B., Gray, V., and Dedhar, S. (1991)
In vitro interaction of a polypeptide homologous to human Ro/ SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin a subunits. Biochemistry 30,9859-9866. (36) Sueyoshi, T., McMullen, B. A., Marnell, L. L., Du Clos, T. W., and Kisiel, W. (1991) A new procedure for the separation of protein Z, prothrombin fragment 1.2 and calreticulin from human plasma. Thromb. Res. 63, 569-575. (37) Uetrecht, J. P. (1988) Mechanism of drug-induced lupus. Chem. Res. Toxicol. 1, 133-143.
