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PubMed1992

Refined definition of the 56K and other autoantigens in the 50-60 kDa region.

Alteration of the acrylamide:bisacrylamide ratio in the SDS-polyacrylamide gel used for Western blotting strongly improved the unambiguous detection of antibodies against 50-60 kDa autoantigens present in autoimmune patient sera. The relative migration of Ro52, the 56K autoantigen and calreticulin increased with reduced acrylamide:bisacrylamide ratios in contrast to that of Ro60, La and Jo-1. These analyses indicated that these six autoantigens correspond to six distinct polypeptides. Further analyses using recombinant calreticulin showed that (i) the 56K autoantigen is neither identical nor related to calreticulin and (ii) calreticulin is not a Ro autoantigen. A series of experiments designed to better characterize the 56K autoantigen showed that (i) the antigen is not detectable in fixed cells, presumably due to masking of the epitopes; (ii) about equal amounts of the antigen were recovered in nuclear and cytoplasmic cell fractions after enucleation of the cells; (iii) the 56K autoantigen is not stably associated with either RNA or other proteins.

Animals↗

PubMed1991

Clarification of the identity of the major M2 autoantigen in primary biliary cirrhosis.

1. In primary biliary cirrhosis, the major M2 autoantigen, reacting with antimitochondrial antibodies in sera from greater than 90% of patients, has been identified as the E2 component of the pyruvate dehydrogenase complex. However, two recent reports suggest that alternative polypeptides may be major autoantigens. 2. The evidence that a 75 kDa subunit of complex I of the respiratory chain is a major autoantigen (Frostell, Mendel-Hartvig, Nelson, Totterman, Bjorkland & Ragan, Scand. J. Immunol. 1988; 28, 157-65) is refuted. The findings of Frostell et al. can be explained by contamination of complex I with the pyruvate dehydrogenase complex, evidence for which is presented here. 3. Inspection of the partial amino acid sequence of an unidentified mitochondrial autoantigen (Muno, Kominami, Ishii, Usui, Saituku, Sakakibara & Namihisa, Hepatology 1990; 11, 16-23) shows that it is the E1 beta-subunit of the pyruvate dehydrogenase complex, previously identified as a major autoantigen, and not a 'new' alternative major autoantigen. 4. These findings substantiate previous work showing that the mitochondrial M2 autoantigens identified so far in primary biliary cirrhosis are all polypeptide components of the pyruvate dehydrogenase complex or the other related 2-oxo acid dehydrogenase complexes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1992

Expression of the 64 kDa/glutamic acid decarboxylase rat islet cell autoantigen is influenced by the rate of insulin secretion.

This study examined the relationship between insulin secretion and expression of the 64 kDa/glutamic acid decarboxylase autoantigen in pancreatic islets. Islets isolated from Wistar rats were cultured for 3 days under different conditions: in 5.5 mmol/l glucose with or without alpha-ketoisocaproic acid or glipizide and in 28 mmol/l glucose with or without diazoxide. The 64 kDa/glutamic acid decarboxylase autoantigen was precipitated from lysates of [35S]-methionine-labelled islets with sera from patients with Type 1 (insulin-dependent) diabetes mellitus and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. In parallel, insulin contents of the islets and the media were determined as well as the rates of glucose-stimulated (pro)insulin biosynthesis. alpha-Ketoisocaproic acid and glipizide were found to stimulate the expression of the 64 kDa/glutamic acid decarboxylase autoantigen and also the rate of insulin secretion. Diazoxide on the other hand reduced the rate of the 64 kDa/glutamic acid decarboxylase autoantigen synthesis in parallel with an inhibition of glucose-stimulated insulin release. Under most of the conditions employed, (pro)insulin biosynthesis was not affected. The correlation found between the rate of insulin release and expression of the 64 kDa/glutamic acid decarboxylase autoantigen might provide an explanation for the earlier observed relationship between the functional demands on the Beta cells and their rate of destruction which may result in diabetes.

Animals↗

PubMed1992

Autoantigenic epitopes of the B polypeptide of Sm small nuclear RNP particles. Identification of regions accessible only within the U1 small nuclear RNP.

OBJECTIVE: To analyze the autoantigenic epitopes of the Sm B polypeptide of the U1 small nuclear RNP (snRNP) using complementary DNA (cDNA) clones. METHODS: Expression of Sm B fusion proteins in lambda phage vectors, immunoblots, immunoprecipitations, and affinity purification of antibodies. RESULTS: Immunoblots using antibodies affinity-purified from B fusion proteins demonstrated that there were cross-reactive epitopes between the B'/B and A polypeptides of the U1 snRNP. Immunoprecipitation assays suggested that there were at least 3 different autoantigenic epitopes on the B polypeptide that could be classified into 2 general groups based upon autoantibody reactivity. The first group of autoantibodies, which bound 2 separate autoantigenic epitopes (BU1-1, BU1-2), participated in immunoprecipitation of the U1 snRNP alone. The second group, which bound the third type of autoantigenic epitope (BSm-1), immunoprecipitated all the abundant Sm snRNPs. CONCLUSION: There is at least 1 region on the B proteins that is accessible to antibodies only within the structure of the U1 snRNP, as well as a region that is accessible on all Sm snRNPs. These data support the notion that the native U1 RNP, perhaps containing B proteins in a different conformation than those found on other Sm snRNPs, may drive the humoral immune response in systemic lupus erythematosus.

Amino Acid Sequence↗

PubMed1975

Ontogensis of spermatozoa autoantigens in guinea pigs.

The ontogenetic appearance of three independant spermatozoa autoantigens (S, P and T) has been studied in guinea pig germinal cells by immunofluorescence and comparison with cytology and histological structures during early maturation of seminiferous tubule cells. The maturation of 120 testes from 60 guinea pigs studied from day 1 to day 50 after birth has shown an evolution in 3 periods. During the first, or negative, period (day 1 to day 25), only spermatogonia (from day 1) and spermatocytes I (from day 16) are present. No significant PAS-positive formations are seen and no autoantigen is detected. During the second, or transitional, period (day 26 to 29), spermatocytes II and spermatids appear as well as paranuclear PAS-positive golgian proacrosomal and acrosomal granules. At the same time, the three autoantigens S, P and T are detected on the same PAS-positive formations with a frequency that increases from day to day. During the third, or positive, period (from day 30) all testes present cells with PAS-positive formation, progressive maturation of acrosomes in spermatids and appearance of spermatozoa (present on day 39) leading to the adult structure of seminiferous tubules. The three autoantigens are constantly present during that period. The simultaneous appearance of the 3 antigens in haploid germinal cells (spermatids and possibly spermatocytes II) as an early expression of cytodifferentiation and their total absence from diploid germinal cells (spermatogonia and spermatocytes I) seem to be of biological significance.

Acrosome↗

PubMed1975

Studies on sperm antigenicity. 2. In vivo and in vitro cellular reactivity in guinea pigs sensitized to homologous and heterologous spermatozoal autoantigens.

In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs of the Rockefeller and Hartley strains, immunized with different preparations of the guinea pig male reproductive tract (RMT) emulsified in complete Freund's adjuvant-H37Ra. The RMT preparations were purified guinea pig spermatozoal autoantigens S, P, and T; whole guinea pig spermatozoa, and extract from epididymal tissue. The cellular sensitivity in vivo was demonstrated by injecting the proper antigen into the skin of the tested animals and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with whole guinea pig spermatozoa cells were inhibited in vitro by the specific antigen, epididymal extract, and autoantigen-T. Autoantigen-S was found to be a weak immunogen. However, the migration of peritoneal exudate cells from guinea pigs sensitized with large amounts of antigen-S was inhibited by whole spermatozoa in vitro. This cross-reactivity revealed the possibility that the immunogenicity of purified autoantigen-S might be connected to its molecular size. According to the immunizing dose of the antigens, testicular lesions of either the aspermatogenic or orchitis type were found in the testes of sensitized guinea pigs. Lesions in the testes of the guinea pigs were not detectable by cross-immunization with heterologous human or rat spermatozoa, although some degree of in vitro cross-reactivity was detected by skin test studies.

Animals↗

PubMed1992

Molecular biology of nuclear autoantigens.

This article provides a historical overview of the application of molecular and immunologic techniques to the analysis of autoantigenic structure and function, as well as to autoantibody recognition of protein and nucleic acid autoantigens. Examples presented here illustrate the role of autoantibodies as tools in the elucidation of the autoimmune components of cellular ribonucleoproteins. In turn, the subsequent molecular dissection of autoantigenic ribonucleoproteins has advanced understanding of autoantibody specificities. The nature of autoantibodies reactive with various proteins and nucleic acids will be the subject of the following articles in this issue. Taken together, these studies of antibody-antigen interactions that arise during the autoimmune response have revealed novel mechanisms of molecular recognition within the RNP autoantigens. These findings are of general importance for understanding basic cellular processes and have contributed to our knowledge of the underlying mechanisms of immunoregulatory abnormalities that arise in autoimmune diseases.

Autoantibodies↗

PubMed1977

Human and guinea-pig cross-reacting spermatozoa autoantigens and autoantibodies.

Cross-reactivity between human and guinea-pig spermatozoa autoanigens has been studied in two systems: (1) guinea-pig anti-autoantigens S, P and T immune sera reacting on human spermatozoa and (2) activity of human anti-sperm sera on guinea-pig spermatozoa. These assays, as well as the results of cross-absorptions in the two systems, revealed a close antigenic relationship between the two species of spermatozoa concerning the guinea-pig autoantigen S. However, there were some differences in its localization and serological manifestations in both species. The guinea-pig autoantigen T bore a more distant, though definite, relationship with human spermatozoa. No clear evidence of cross-reactivity between the two species was found for autoantigen P. At variance with autoimmune (or isoimmune) human antibodies, 'natural' human antibodies active on guinea-pig spermatozoa were absorbed by guinea-pig spleen cells.

Animals↗

PubMed1979

Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.

Animals↗

PubMed1991

Reverse immunoaffinity chromatography: application to the purification of the 60- to 90-kDa gastric parietal cell autoantigen associated with autoimmune gastritis.

Previous attempts to purify autoantigens by affinity chromatography using the total IgG from autoantibody-positive sera have not been very successful. We describe here a novel method using parietal cell autoantibodies, isolated on a crude autoantigen immunoabsorbent column, for the purification of the 60- to 90-kDa gastric parietal cell antigen targeted in autoimmune gastritis. The 60- to 90-kDa parietal cell antigen was first enriched 2.5-fold from a 0.5% Triton X-100 extract of total pig gastric mucosal membranes by DEAE-Sepharose 4B chromatography. The fraction containing the 60- to 90-kDa antigen was directly coupled to CNBr-activated Sepharose 4B to produce an immunoadsorbent column for the "reverse" purification of the specific parietal cell autoantibodies from pooled gastric parietal cell autoantibody-positive human sera. A 0.5% Triton X-100 extract of pig gastric mucosal membranes was initially precleared by sequential passage through Protein A-Sepharose 4B and normal human Ig-Sepharose 4B columns. The 60- to 90-kDa gastric parietal cell antigen (approximately 10 micrograms) was then purified from this precleared Triton X-100 membrane extract by affinity chromatography on a 1-ml column of purified autoantibody-Sepharose 4B, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophonesis. This method, incorporating a "reverse" immunoaffinity chromatography step, has general applicability for the purification of tissue autoantigens using highly specific autoantibodies present in the sera of humans and experimental animals with a variety of other autoimmune diseases.

Animals↗

PubMed1975

Characterization of autoantigenic sites on isolated dog heart mitochondria.

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).

Animals↗

PubMed1992

Centromere autoantigens are associated with the nucleolus.

Because of their importance as target antigens in scleroderma and since all other major autoantigens in scleroderma can be localized to the interphase nucleolus, we were interested in a further investigation of the potential relationship between interphase centromeres and the nucleolus. Using human anticentromere autoantibodies (ACA) from patients with the CREST form of scleroderma as probes in indirect immunofluorescence microscopy, we observed nonrandom interphase "clumping" of centromeres in a distribution suggestive of nucleoli. By double-label immunofluorescence comparing the localization of centromeres to nucleolar proteins Ki-67, fibrillarin, or protein B23 (nucleophosmin), interphase centromeres appeared to be localized around and within nucleoli. A number of different ACA sera were tested on HEp-2, HeLa, PtK2, Indian muntjac, 3T3, and NRK cells, all with identical results indicating colocalization between centromeres and nucleoli. Immunoelectron microscopy revealed that interphase centromeres were distributed free in the nucleoplasm, in contact with the nuclear envelope, in contact with and on the periphery of nucleoli, and totally embedded within the confines of the nucleolus itself. Interestingly, actinomycin D treatment dissociated centromeres from localization within the segregated nucleolus. To determine if interphase centromeres were integral components of nucleoli, nucleoli were isolated according to classical methods. By double-label immunofluorescence, immunoelectron microscopy, and Western blotting, it was demonstrated that centromere autoantigens copurified with isolated nucleoli. These studies offer proof that some interphase centromeres can be associated with, and may even be considered part of, the interphase nucleolus. Furthermore, all of the major autoantigens in scleroderma can now be localized to the nucleolus.

Autoantigens↗

PubMed1992

21-Hydroxylase, a major autoantigen in idiopathic Addison's disease.

Sera from patients with idiopathic Addison's disease commonly react with the zona glomerulosa of adrenal cortex. We used high-resolution western blot analysis of an adrenal microsomal fraction to investigate the target of these antibodies. A protein with an apparent molecular weight of 54 kDa was recognised as the common and major component. Sera identifying this autoantigen (from 12 of 16 patients) also showed strong immunofluorescence staining of a steroid-producing human adrenal adrenocortical cell line, NCI-H295. On application of antisera specific for different cytochrome P450 steroidogenic enzymes (side-chain cleavage enzyme, 21-hydroxylase, 17 alpha-hydroxylase, 11 beta-hydroxylase) the mobility of the 54 kDa protein in western blots corresponded to that of 21-hydroxylase. This parallel behaviour was confirmed by immunoprecipitation, electrophoresis, and autoradiography with the various sera and 35S-methionine-labelled NCI-H295 cell lysates. Preabsorptions of 35S-methionine-labelled cell lysates with the antiserum to 21-hydroxylase, but not with the other enzyme antisera, abolished precipitation of the 54 kDa autoantigen with the patient sera. These results indicate that 21-hydroxylase (P450c21), prominent in the zona glomerulosa of the adrenal cortex, is a major autoantigen in idiopathic Addison's disease.

Addison Disease↗

PubMed1992

Secretory granule autoantigen in insulin-dependent diabetes mellitus is related to 62 kDa heat-shock protein (hsp60).

Recent studies in NOD mice suggest that cellular and humoral responses against beta cell protein(s) cross-reactive with mycobacterial heat-shock protein, hsp60, are implicated in the development of autoimmune diabetes. However, this putative, hsp60-related autoantigen has not yet been identified nor have the preceding events triggering the autoimmunity against it. Our recent studies show that antibodies to the mammalian hsp60 bind specifically to the 62 kDa protein located to insulin secretory granules and mitochondria of pancreatic beta cells of healthy mice [1]. In islets of prediabetic NOD mice affected by insulitis, the cellular distribution of this hsp60-related antigen was found to be altered. In the present report, we have examined whether this endogenous hsp60-related protein of secretory granules serves as an autoantigen in type I diabetes. The results of Western blot analysis indicate that diabetic mice sera show reactivity to a 62 kDa islet cell antigen. The NOD mice sera that were positive in detection of the 62 kDa islet cell antigen were also able to recognize the recombinant human hsp60. Immunogold electron microscopy revealed that diabetic NOD mouse sera, cross-reactive to human recombinant hsp60, recognize the antigen located in secretory granules of beta cells. Double-immunogold labelling demonstrated that antigens recognized by both diabetic NOD mice sera and monoclonal hsp60 antibodies co-localized in the same secretory granules of beta cells. Preincubation of islet cell sections with one type of antibody blocks subsequent binding of the other, indicating that epitopes recognized by both antisera on these proteins are shared. Moreover, preadsorption of diabetic sera with the recombinant human hsp60 abolished labelling of secretory granules. These results indicate that the hsp60-related protein of beta cell secretory granules is an autoantigen in type I diabetes in NOD mice.

Animals↗

PubMed1992

Characterization and epitope mapping of human monoclonal antibodies to PDC-E2, the immunodominant autoantigen of primary biliary cirrhosis.

Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping of PDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five monoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fc), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial autoantigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2.

Animals↗

PubMed1992

Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.

The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.

Antigens, Nuclear↗

PubMed1978

Immunological T-cell memory in the in vitro-induced experimental autoimmune orchitis: specificity of the reaction and tissue distribution of the autoantigens.

Immunological memory has been induced in vitro against testicular autoantigens by priming normal rat T lymphocytes against autologous testis cells, and by permitting the isolated blast cells to revert back to small secondary lymphocytes (secondary EAO cells) in the absence of the priming antigen. The secondary EAO cells vigorously respond in a secondary response when reconfronted with syngeneic testis or lymphoid cells. Their responsiveness to nonself stimulator cells is, however, reduced. Secondary cells derived from concanavalin A-stimulated blasts, do not show that pattern of specificity. The specificity of the secondary EAO cells is definite, and cannot be affected by further culture on allogeneic fibroblasts, which are antigenic for unprimed T lymphocytes. At least part of the autoantigens are determined by the major histocompatibility gene complex (MHC). Factors provided by the culture system do not appear to determine the specificity of this reaction. Only minor cell populations can restimulate secondary EAO cells. One of these populations is presumably phage-like cells within the lymphoid populations can elicit a secondary EAO response. Thus, the autoantigens relevant in the secondary EAO response are either MHC antigens restricted to these testicular and lymphoid subpopulations, or MHC antigens recognized in conjunction with organ-specific non-MHC determinants.

Animals↗

PubMed1991

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.

Adenosine Triphosphatases↗