Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes

Acta histochem. (lena) 96. 51-62 (1994) Gustav Fischer Verlag lena' Stuttgart· New York

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Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes Wolfgang Kreisel', Heika Hildebrandt' , Emil Schiltz", Gabriele Kohler 3 , Cornelia Spamer' , Christine Dietz', Wolfgang Mossner', and Claus Heilmann' I Medizinische Universitiits-Klinik. Abteilung Gastroenterologie und Hepatologie, Hugstetter Str. 55. 2Institut fiir Organische Chemie und Biochemie and 3 Pathologisches Institut. Albert-Ludwigs-Universitiit. Freiburg. 79106 Freiburg. Germany

Accepted 31 October 1993

Summary We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructural localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium binding protein calsequestrin by immunoabsorption on isolated rat liver hsp60. As shown by partial N-terminal amino acid sequence analysis of immunoprecipitated proteins AB 121 contained reactivities against hsp60. calsequestrin and the glycoprotein fetuin. In rat heart AB 121 recognized calsequestrin and hsp60. In human and rat liver the only reacting protein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody was used for immuno-gold electron microscopical localization of hsp60: in myocardiocytes and hepatocytes of the rat strong labelling was obtained exclusevely in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visualized by immuno-gold electron microscopy offers the possibility to study the expression of this protein in the liver and in other organs. Possible clinical applications of these studies are discussed. since hsp60 could be a target antigen of autoantibodies in diseases such as autoimmune hepatitis. primary sclerosing cholangitis or primary biliary cirrhosis.

Key words: heat shock protein 60 - calsequestrin - fetuin microscopy - mitochondria - autoimmune liver diseases

immuno gold electron

Introduction In several conditions such as exposure to elevated temperature, hypoxia, oxidizing agents, infections. in pro- and eukaryotic cells a set of proteins, the stress proteins or heat shock proteins (hsps) is synthesized (Burdon, 1986; Schlesinger, 1990; Burel, 1992). The expression of a subset of hsps is also induced during differentiation or embryogenesis. Hsps of all Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 68th birthday. Correspondence to: W. Kreisel

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species ranging from bacteria to mammalians contain a very high proportion of sequence homologies and belong to the most conserved proteins. Generally, hsps function as molecular chaperones, mediating folding, assembly or translocation of other proteins across intracellular membranes and playa role in protein degradation (Ellis, 1989; Gupta, 1990). Associations between steroid hormone receptor activity or oncogene products and hsps are described (lkawa, 1992). Furthermore hsps play a role in bacterial infections (Garbe, 1992), immunity against bacteria and in autoimmunity (Kaufmann, 1990; Kaufmann, 1992; Mollenhauer, 1992; Yang, 1992). Investigating the specificity of a polyclonal antiserum originally raised against dog heart calsequestrin (es) we found that it strongly reacted with 1. es, 2. hsp60, 3. fetuin. In liver and cultured hepatocytes the only immunoreactive protein is hsp60, a protein of the mitochondrial matrix. This offers the possibility to study the expression of hsp60 in the liver under normal and pathological conditions such as acute and chronic liver diseases. Of special interest are autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. In these chronic liver diseases mediated by autoimmunological processes hsps could function as autoantigens.

Materials and Methods ChoniUl/.I'. All chemica" were of analytical grade. They were purchased from Sigma (Deisenhofen, Germany) or Roth (Karlsruhe, Germany). Nitrocellulose membranes were from Hoefer Scientific Instruments (Minnesota, LISA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad (Mlinchen, Germany). Materials for electron microscopy were supplied by Plano (Marburg, Germany) or Sciences Services (Mlinchen, Germany).

Protein "eterlllination. Pmtein was determined according to the method of Lowry (I YSI) using human serum albumin as a standard. SDS PAGE. Sodium dndecyl sulfate polyacrylamide gel electrophoresis was performed according tn Laemmli (1 Y7()). Proteins were stained with Coomassie Brilliant Blue R-2S0 or Pon,eau Red or Stains All. Relative molecular weights were determined using calibration proteim.

Western illllllll/w NOlling. immunoblotting was carried out as described by Towbin (1 Y79) using commercially available horseradish peroxidase-conjugated anti-rabbit IgG as a second antibody. Immunoreactive proteins were visualized with the peroxidase reaction. Iso/ation or lIIitoc//(Il1dria. Mitochondria were isolated fmm rat heart and liver according to a procedure published by Brdiczka (1%9).

Preparation or errtl1mcyt!' ('\'10/'/0.1'111. Blood of male Wistar rats containing heparin for prevention of clotting was centrifuged at 1SOO X g for 1() min. After washing two times with PBS the pelleted cells were diluted with PBS (1: 2) and underlayed with Ficoll. After 30 min centrifugation at 1500xg the pelleted cells (mainly erythrocytes) were washed with PBS. Thereafter an equal volume of H20 was added for cell lysis. The memhranes 0]' erythrncytes were pcllcted and the supernatant was taken for further studies. ISO/Iltioll of dog h('ur/ m/.I(,(/IIC1trili. 100 g of dog heart, previously perfused in situ with ice-cold O.Y% Nae!, were minced with scissors. Minced tissue was suspended in 400 ml of a solution containing O. I M KH,PO.j, 2.66 M (NH.j)2S0.j. I mM EDTA, S mM 2-mercaptoethanol, 1.5 mg/l leupcptin, 3.3 mg/l aprotinin, 0.1 mM rhenylmethylsulfonyl fluoride, 0.4 mM benzamidine, pH 7.1 (adjusted with NaOH). The

Electron microscopical detection of heat shock protein 60

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suspension was homogenized at °C for 2 x 30 sec in an Omnimixer (Sorval!, Newton, USA) with rheostat setting at 10, All subsequent preparative steps including differential centrifugation and ammonium sulfate precipitation were carried out following the method published by Campbell (1983) and Slupsky (1987). The final ammonium sulfate precipitate was dissolved in 0, I M potassium phosphate buffer (pH 7, I) containing I mM EDT A and dialyzed against the same buffer for 16 h followed by 4 h dialysis against 50 mM NaC!' 0,1 M potassium phosphate (pH 7,11, I mM EDTA, The suspension was centrifuged for 10min at 9000Xg, The supernatant was applied to a 5 x 10 cm DEAE-cellulo,,~ column equilibrated with 50 mM NaC!' 0, 1M potassium phosphate (pH 7,1), I mM EDTA, 5mM 2-mercaptoethanoL The column was extensively washed with equilibration buffer. Protein fractions (5 ml!fraction) were eluted with a linear gradient of 0,05 to 1,0 M NaCI containing 5 mM 2-mercaptoethanoL Each fraction was analyzed for sodium concentration by flame photometry and for protein patterns by SDS-PAGE, Fractions enriched in CS were identified by staining gels with Stains AIL that stains this protein intensively blue, Peak fractions were pooled and then concentrated and desalted by ultrafiltration using a high-pressure Amicon filtration device equipped with a PM 10 filter. Iso/ution of rat /ilW hsp60, Rat liver mitochondria were isolated as described below, Mitochondrial proteins were separated on PAGE and transferred to a nitrocellulose membrane, After localization of hsp60 by the immunoperoxidase reaction hsp60 was cut out from the gel and used for purification of anti-hsp60 reactivity, Preparation of antiserulIl containing £lIl1i-hsp60 reactivity, Fractions from DEAE-cellulose chromatography containing highly enriched dog heart CS were separated on SDS-PAGE. Gels were stained for proteins with Coomassie Brilliant Blue R-250, Protein bands containing the antigen were cut out of the gel and washed :3 x 10 min with destilled water. The gel pieces containing appmximately O,lmg of the antigen were extensively emulsified in 1,2 ml of complete Freund's adjuvant The antigen was injected subcutaneously in four sites at the back of New Zealand White male rabbits, followed by booster injections at three weeks intervals, A small volume of preil11mune serum of each rabbit was fmzen and stored at -20°C, Two weeks after the third injection the sera were immunoreactive with the antigen at a dilution of I : 4000 (Western immunoblot analysis), For the conti'll!' preimmune sera were used which did not result in any detectable iml11unoreaction with the antigen, From the antisera the IgG fraction was isolated by ammonium sulfate precipitation and affinity chromatography on protein A-Sepharose according to standard procedures, The IgG fraction containing the anti-CS antibody was termed AB 121, From AB 121 the anti-hsp60 IgG was purified by immunoabsorption on isolated rat liver hsp60 transferred to nitrocellulose and desorption by 0,2 M glycine buffer pH 2,), Ana/ysis o( N-tf'rmina/ lImino acid sn/lIenC(', N-Terminal sequencing of the electroblotted proteins was performed in an automated protein sequencer equipped with on-line analysis of the phenylthiohydantoin amino acids (Models 477A and 120A, Applied Biosystems Inc" Foster City, USA). immllllOeiectl'On microscopy, Colloidal gold solutions were prepared according to Horisberger (1977), 15 nm gold particles were bound to protein A as described by Slot (1985), Rat heart and liver tissues were fixed using a method modified according to Karnovsky (1965): After starvation for 24 hours, the organs were perfused for 20 min with 2'1<, paraformaldehyde and 0, I % glutaraldehyde in PBS, pH 7,4, Thereafter they were excised and stored for 4 hours in the same solution, followed by a 12 hours incubation with 0,05 M NH.jCI in PBS, Embedding was done in Lowicryl K4M by progressive temperature lowering down to - 20°C: polymerization was performed under UV light in the cold (Kellenberger, 1965), Thin sections were applied to nickel grids, After incubation with the anti-hsp60 IgG (diluted I : 10 with PBS) labelling with 15 nm gold particles bound to protein A was done (Roth, 1983: Roth, 1984), The sections were contrasted with uranyl acetate and lead citrate according to standard procedures,

Results As shown in Fig, I, the antibody AB 121 binds to several proteins distinguishable by their relative molecular weight: Lanes I and 2 demonstrate the reaction with authentic isolated dog heart CS and rat heart CS, respectively, In the homogenate of rat heart

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Fig. l. Western immunoblot anal ysis of proteins from rat heart. liver and ery throcytes using A B 1 2 1: lane I : Dog heart calsequestri n. lane 2: Rat heart calsequestrin . lane 3: Rat heart homogenate. lane 4: Rat li ver homogenate. lane 5: Rat heart mitochondri a. lane 6: Rat li ver mitochondri a. lane S: Rat erythrocyte cytoplasm. lane 7: Western immunoblot analysi s of proteins fro m rat liver mitochondria using im munoabsorbed anti-hsp60 JgG. <).5 % SDS-PAGE. proteins transferred to nitroce llul ose. visual ization of immunoreacti ve prote ins using pem xidase-coupled secondary antibody.

two reactive polypeptides are fo und with Mr of 6 1000 an 56000 (lane 3 ). The latter corresponds to rat heart CS . From lane 5 it can b e concluded that the 6 1 kDa protein is of mitochondrial origin . Rat liver contains onl y one polypeptide which reacts with the antibody. Its M, amounts to 6 1000 (lane 4) . As shown i n lane 6. it is localized in mitochondria . This s uggests a n di entity with the 6 1kDa prote in from heart mitochondria. Immunoabsorbed ant i-hsp60 IgG strongly reacts with hsp60 from r at li ver mitochondria (l ane 7). Surprisingly, two polypeptides of rat eryth roc yte cytoplasm with Mr of 6 1000 and 58000 are recogni 7.ed b y the antibody (lane 8).

Electron microscopical detection of heat shock protein 60

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Table I. N-terminal amino acid sequence of the immunoreactive proteins Protein

N-terminal amino acid sequence

rat Ii ver hsp60 (Peralta (1990))

AKDYKFGADAR

61 kDa protein of rat liver mitochondria (this study)

AKDYKFGADA

fetuin (Rauth (1990))

APQGAGLGFR

ELACDDPETE

61 kDa protein of erythrocytes (this study)

APQGAGLGFR

ELA

58 kDA protein of erythrocytes (this study)

APQG

ALMLQGYDL

LADAYAY

XALMLQGY

HYALI

For the identification of the immunoreactive proteins partial N-terminal amino acid sequence analysis was performed (Table I); 18 amino acids of the N-terminus of the mitochondrial 61 kDa protein (isolated from rat liver mitochondria) were sequenced (one amino acid could not be identified). They show a 100% identity with the published sequence for rat hsp60. The amino acid sequence of the first 13 amino acids of the erythrocytic 61 kDa protein completely correspond to the published sequence of fetuin, a glycoprotein of rat plasma. From the 58 kDa protein of erythrocytes only a four amino acid sequence was obtained. It is identical to that of the erythrocytic 61 kDa protein. Therefore, we assume that both proteins represent differently glycosylated forms of fetuin. For the further investigation of the localization and expression of hsp60 in different cell types a monospecific polyclonal anti-hsp60 IgG was prepared from AB 121 by immunoabsorption on hsp60 isolated from rat liver mitochondria. Lane 7 in Fig. 1 shows the Western immunoblot analysis of rat liver mitochondrial proteins using anti-hsp60 IgG. Using anti-hsp60 IgG the ultrastructural localization of hsp60 was analyzed: As shown in Fig. 2 a, in rat heart only mitochondria are labelled with gold particles. Whether membranes or the matrix are labelled cannot be distinguished. No other structure is labelled by the antibody; no label is detectable in the nucleus. Fig. 2 b shows the background labelling using preimmune rabbit IgG. These results fit well to the assumption, that hsp60 is exclusively localized in mitochondria. Figs . .) shows corresponding experiments with hepatocytes. Liver ultrastructure can be preserved if the rats were starved for 24 hours before sacrificing. The labelling of mitochondria can be clearly seen (Fig . .) a). Evidently nuclei do not contain immunoreactive protein. Again, the control experiments using preimmune IgG yield only a weak scattered labelling of the cells without any preference of distinct structures (Fig. 3 b). As demonstrated in Fig. 4, AB 121 (which contains immunoreactivity against calsequestrin, hsp60 and fetuin) strongly labels the erythrocyte cytoplasm. In combination with the results shown in Fig. I this proves the existence of fetuin within the erythrocyte. The nucleus of a cell surrounding the blood vessel (probably a pericyte) is not immunodecorated.

Discussion In the present paper we characterize a highly reactive polyclonal antibody against hsp60 and describe a first application for immunogold electron microscopical localization studies

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Fig. 2. Detection 01 hsp()() in rat twart hy inlmuno-t!0ld eicctmnlllicnl\copy. a. Labeling 01 an ultrathin section oi' rat heart using anti-hsp hU IgG. Only mitochondria are labelled. The inset shows the same section with lower magnii'ication. h. Control using preimlllune rahhit serum.

Electron microscopical detection of heat shock protein 60

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fig . .3. Detection of hsp60 in rat hepatocytes hy illllllul1o-gold electron mic[nscupy. a. Ultrathin sectiun of rat liver labelled with anti-hsp I1'G. Only mitochondria arc labelled. The inset shows the same section with lower magnirication. h. Control using preillllllulle rabbit seruill.

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Fig. 4. Immuno-gold electron microscopy of a small blood vessel using AB 121. Mitochondria of a endothelial cell slIrrouding the blood vessel and the cytoplasm of an erythrocyte are labelled with gold particles.

of this protein. Originally the aim of our study was to induce a polyclonal antibody against calsequestrin (CS). This protein is a constituent of the luminal face of the terminal cisternae of sarcoplasmic reticulum. It is suggested that CS, which binds calcium at high capacity but with moderate affinity, functions as a calcium storage protein within the lumen of the SR and might be also involved in the mechanism of calcium release from the SR. In mammalians two forms can be distinguished: I. the heart muscle type occurring in heart muscle, smooth muscles and slow-twitch skeletal muscle and 2. the fast-twitch skeletal muscle type (Campbell. 1983 ~ Wuytack, 1987; Scott, 1988; Fliegel. 1989 a, b). Mammal ian calsequestrins share considerable sequence homologies. However, the CSs from different species show slight variations in primary structure. relative molecular mass, and degree of glycosylation. As expected, nativc dog heart calsequestrin is effectively immunoprecipitated hy AB 121. Rat heart CS serves as a target for this antihody also.

Electron microscopical detection of heat shock protein 60

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Surprisingly, our studies revealed reacting proteins in heart, liver and erythrocytes with relative molecular masses of 61000 and 58000, respectively. Subcellular fractionation showed that the 61 kDa protein of heart and liver is localized in mitochondria. In liver, this protein is the only one reacting with the antibody. In erythrocytes, both reacting proteins are recovered from the soluble cytosolic fraction. By partial N-terminal amino acid sequence analysis the proteins could be identified: the 61 kDa protein of mitochondrial origin is identical to heat shock protein hsp60 (Peralta, 1990); the 61 kDa protein found in erythrocytes corresponds to the glycoprotein fetuin (Rauth, 1992); from the 58 kDa protein only the first four amino acids could be sequenced. As they correspond to the N-terminus of the erythrocytic 61 kDa protein we assume, that this polypeptide represents an isoform of fetuin (Hayase, 1992). According to the published amino acid sequences CS, hsp60 and fetuin do not share common sequences nor could common motifs be found, which might explain a crossreaction. However, conformational epitopes are not excluded. Considering the method for isolation of dog heart CS it is possible that traces of hsp60 and fetuin would have contaminated the isolated protein used for immunisation. Another fact to be taken into account is that fetuin can be detected in rat plasma but not in serum. So traces of fibrin clots which may contain fetuin could have been present in the perfused hearts that have been the source for dog heart CS. Since hsp60 is an unglycosylated protein, a reactivity of the antibody against common glycan structures of the three antigens can be excluded. A strong argument against an antigenetical relationship of calsequestrin, hsp60 and fetuin is the fact that we were able to separate the reactivities against the respective proteins by immunoabsorption of AB 121 on the purified proteins. In this way we obtained highly reactive poly clonal IgG against CS, hsp60 and fetuin, respectively. Our studies on CS and fetuin will be described separately. Here we present an immunogold electron microscopical localization study as a first application of the anti-hsp60 IgG. In heart muscle cells only mitochondria have the immunoreactive protein. No other organells are labelled with gold particles. This corresponds to the immuno-gold labelling experiments with hepatocytes. Again, the mitochondria are labelled with gold particles. The strong labelling of mitochondria of hepatocytes ill situ with a very low background suggests that no other structure contains hsp60. Even in a cell surrounding a blood vessel (possibility a pericyte) the small mitochondria were labelled underlining the fact that hsp60 is a nearly ubiquitous mitochondrial protein. The localization of hsp60 in mitochondrial has been derived from biochemical methods so far. The present paper adds the first ultrastructural evidence for a localization of hsp60 exclusively in mitochondria. Recently, it was confirmed by immunogold electron microscopy that DnaK, a member of the hsp70 protein family, is mainly a cytosolic protein; only part of this protein is membrane-associated (Bukau, 1993). Using AB 121. which also shows reactivity against CS, only hsp60 was found as a immunoreactive protein in rat liver homogenate. This is of special interest. since several authors postulated the existence of calciosomes in hepatocytes. These calcium-storing vesicles should contain CS or a related protein (Hashimoto, 1988; Volpe, 1988; Krause, 1989). However. this assumption has been questioned by others (Van, 1989). At least using Western immunoblot analysis we could not find CS in the liver. By immunogold electron microscopical analysis using AB 121 no extramitochondrial structures reminescent of calciosomes were detected (data not shown in this manuscript). We intend to study the distribution of hsp60 in the liver lobule by immunohistochemistry using AB 121. Because of the localization of hsp60 in mitochondria it will be interesting to investigate whether its expression parallels the uneven distribution of mitochondria over the lobule. In the periportal zone mitochondria comprise approximately 20')\, of the hepatocyte volume and in the perivenous zone only 12 % (Wimmer and Pette 1979; AsadaKubota, 1982; KanaL 1986). This is reflected by the preferential localization of OT

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consuming metabolic pathways such as gluconeogenesis, fatty acid oxidation or urea synthesis in the peripheral zone of the lobule, whereas glycolysis, lipogenesis, ketogenesis, glutamine synthesis and metabolism of xenobiotics are concentrated to the perivenous hepatocytes (Katz, 1989; Jungermann, 1982; Haussinger, 1983). Accordingly there is a gradient in O2 saturation from the periphery to the center of the liver lobule (Quistorff, 1986; Matsumura, 1983; Baraona, 1983). Since hypoxia is one of the stress factors leading to increased expression of hsps one could speculate that a decreased perivenous O 2 concentration could lead to an enhanced expression of hsp60 in perivenous hepatocytes. During bacterial or mycobacterial infections in humans T-cells are induced which react with bacterial and human hsp60 (Garbe, 1992; Mollenhauer, 1992). This may in some circumstances lead to autoimmunity, whereby human hsp60, which is known to contain more than 50% sequence homologies to prokaryontic hsp60 or hsp60 analogues such as mycobacterial hsp65, can be the target antigen (Kaufmann, 1990; Kaufmann, 1992; Yang, 1992). Additionally, sequence homologies were found between human hsp60 and a number of defined autoantigens (Jones, 1993) in a variety of autoimmune diseases: e.g. cytochrome P450 (Manns, 1990) in chronic active hepatitis, dihydrolipoamide dehydrogenase (Coppel. 1989) in primary biliary cirrhosis or 17 (,(-hydroxylase in Addison's disease (Krohn, 1992). These findings fit well to the hypothesis that autoimmune processes are induced by molecular mimicry between microbial or viral peptides presented by antigenpresenting cells and self peptides presented inappropriately on a target tissue (Baum, 19S13). The antibody AB 121 cross-reacts with human hsp60 (data not shown). This opens the possibility to study the expression of hsp60 in normal and diseased liver. Of special interest would be conditions such as autoimmune hepatitis, primary biliary cirrhosis or primary sclerosing cholangitis. Ultrastructural localization studies using the method and the antibody described here could help to answer the question if an altered expression of hsp60 or its aberrant localization in the cellular surface membrane may trigger or perpetuate an autoimmune process.

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Fliegel L, Burns K. Wlasichuk K. and Michalk M (19X9) Peripheral membrane proteins of sarcoplasmic and enuoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences. Biochem Cell Bioi 67: 696-702 Fliegel L, Leberer E. Green NM. and MacLennan DH (19X9) The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twich soleus muscle. FEBS Lett 242: 297 - 300 Garbe TR (1992) Heat shock proteins and infection: Interaction of pathogen and host. Experientia 48: 635-639 Gupta RS i 1990) Mitochondria. molecular chaperone proteins and the ill I'j\'() assemhly of microtubules. TlBS 15: +IS-+IX Haussinger 0 (19X31 Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intracellular glutamine cycle during ureagenesis in perfuseu liver. Eur J Biochem 133: 269-275 Hashimoto S. Bruno B. Lew DP. Pozzan T. Volpe P. anu Meldolesi J (198X) Immunocytochemistry of ca\ciosomes in liver and pancreas. J Cell Bioi 107: 2S23-2531 Hayase T. Rice K. Dziegielewska M. Kuhlenschmidt M. Reilly T. and Lee YC (1992) Comparison of Nglycosides of i'etuins from uifferent species and human o2-HS-glycoprotein. Biochemistry 31: 4915-4921 HllI'isberger M. and Rosset J (1977) Colloidal gnld. a useful marker fnr transmission and scanning electron microscopy . .f Histochcm Cytnchcm 25: 2<)5-3()5 Ikawa S. and Weinberg RA (19921 An interaction between p21 "" and heat shock protein hsp60. a chaperonin. Proc Natl Acad Sci USA 89: 2012-201n Jones DB. Coulsnn AFW. and Dull GO (I <)931 Sequence homologies between hsp60 and autoantigens. Immunology Tnday 14: IIS-IIX Jungermann K. anu Katz N (I<)X21 Functional hepatocellular heterogeneity. Hepatology 2: 3X5-3<)5 Kanai K. Kanamura S. and Watanabe J (19X61 Peri- anu postnatal development of heterogeneity in the amounts of endoplasmic reticulum. Am J Anat 175: +71-+XO Karnovsky MJ (1965) A fnnnaidehyue-giutaraidehyde fixative of high osmolality for use in electron microscopy. J Cell Bioi 27: U7A-13XA Katz NR (1989) Method, for the study of liver cell heterogeneity. Histochemical J 21: 517-529 Kaufmann SHE (1990) Heat shock proteins and the immune response. Immunology Today 11: 129-136 Kaufmann SHE (1992) The cellular immune response tll heat shock proteins. Experientia 48: 640-643 Kellenherger E. Carlemalm E. Villiger W. Roth]. and Garavito RM (1980) Low uenaturation embedding for electron miel'llscopy of thin sectinns. Chemische Werke Lowi. Waldkraiburg. Germany Krause KH. Pittet D. Vllipe P. Pozzan T. Me\dolesi 1. and Lew DP (1989) Caiciosome. a sarcoplasmic reticulum-like organelle involved in intracellular Ca 2+ -handling by non-mu,c\e cells: Studies in human neutmphils and HL-60 cells. Cell Calcium 10: 351- 361 Krohn K. Uibo R. Aavik E. Petersnn P. and Sarilahti K (I <)<)2; Identification by molecular cloning of an antvantigen associated Addison's disease as steroid 17 alpha-hydroxylase. Lancet 339: 770-773 Laemmli UK (I <)701 Cleavage of structural proteins during the assembly of the head of the bacteriophage T •. Nature 227: hHO-6XS Lowry OH. Rosenbrough NJ. Farr AL. and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Bioi Chem 193: 265-275 Manns MP. Griffin KJ. Quattrochi LC, Sacher M. Thaler H. Tuckey RH. and Johnson EF (1990) Identification nf cytochrome P4S0lA2 as a human autoantigen. Arch Biochem Biophys 280: 229- 232 Matsumura T. and Thurman RG ( 1983) Measuring rates of O 2 uptake in periportal and pericentral regions of the liver lobule: Stop-now experiments with perfused liver. Am J Physiol 244: G656-G659 Mollenhauer J. and Schulmeister A (1992) The humoral response to heat shock proteins. Experientia 48: 644-649 Peralta D. Hartman OJ. McIntosh AM. Hoogenraad NJ. and Hoi PB (1990) C-DNA and deduced amino acid sequence of rat liver prehsp60 (chaperonin-60). Nucleic Acids Res 18: 7162 QuistorlT B. and Chance B (1986) Redox scanning in the study of metabolic zonation of liver. In: Thurman RG. Kauffman Fe. and Jungermann K (Eds) Regulation of hepatic metabolism. Plenum Press. New YorkLondon. pp I XS - 207 Rauth G. Paschke O. Fink E. Eulitz M. Tippmer S. Kellerer M. Haring H-U. Nawratil P. Haasemann M. Jahnen-Dechent W. and Muller-Esterl W (1992) The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor. Eur J Biochem 204: 523-529 Roth J (1<)8.1) The colloiual gold marker system for light and electron microscopy cytochemistry. In: Bullock GR. and Petrusz P (Ed.q Techniques in immunocytochemistry. Vol. II. Academic Pre.s". London. pp 217-28+

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