Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

Biochemical analysis of the receptor for ubiquitin-like polypeptide.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-beta, an isoform of the MNSF, has been isolated and characterized. MNSF-beta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to unstimulated D.10 cells. Detergent-extracted membrane proteins were applied to an immobilized Ubi-L column. SDS-polyacrylamide gel electrophoresis of eluted fraction revealed a band of Mr = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of Mr = 90,000 was visualized by immunoprecipitation of 125I-Ubi-L cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide) antibody, indicative of the association of this protein with the Ubi-L receptor complex. Amino acid sequence analysis of the 82-kDa polypeptide revealed that the Ubi-L receptor may be a member of a cytokine receptor family.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Abstract Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-β-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica . The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-β-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-β-glucan synthase activity.     

Characterization of a murine monoclonal antibody that detects a C-terminal fragment of the raf oncogene product

A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector. Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines. Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts. Parallel experiments with polyvalent antiserum prepared against E. coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously. Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining. This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein. It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity

A partially purified preparation of human erythrocyte protein 4.9, consisting of 48-, 52-, and 55-kilodalton polypeptides, is capable of bundling rabbit muscle actin in vitro (Siegel, D. L., and Branton, D. (1985) J. Cell Biol. 100, 775-785). Purification schemes, peptide mapping, antibody cross-reactivity, and chemical cross-linking techniques show that the 48- and 52-kDa polypeptides are sequence-related phosphorylated components, whereas the 55-kDa polypeptide is not. Purified protein 4.9 (dematin), consisting of 48- and 52-kDa polypeptides, effectively bundles actin in vitro; under similar conditions, the isolated 55-kDa polypeptide does not bundle actin. In fact, when added back to purified dematin, fractions containing the 55-kDa polypeptide can completely abolish dematin's actin-bundling activity. The basis for this inhibitory activity is an endogenous protein kinase that phosporylates both the 48- and 52-kDa isoforms of dematin, thus abolishing dematin's actin-bundling activity (Husain-Chishti, A., Levin, A., and Branton, D. (1988) Nature 334, 718-721). Although the endogenous kinase often co-purifies with the 55-kDa polypeptide, it can be separated from the 55-kDa polypeptide and has the characteristics of a catalytic subunit of a cyclic AMP-dependent protein kinase.     

Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

Protein components of the nonactivated glucocorticoid receptor.

The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.     

Isolation and partial characterization of the human erythrocyte band 7 integral membrane protein.

Monoclonal antibodies to the Mr 31,000 major integral membrane protein of the human erythrocyte band 7 region were used to identify the corresponding polypeptide chain and epitope-carrying fragments on immunoblots. Analysis of the erythrocyte membrane, membrane fractions, and cytosol revealed that the Mr 31,000 band 7 integral membrane protein is unique and not related to any of the other water-soluble or membrane-bound band 7 components. Cross-reacting proteins were identified in the membranes of other mammalian erythrocytes and in cell lines of epithelial and lymphoid origin. Proteolytic digestion of intact human erythrocytes or erythrocyte membranes demonstrated that the band 7 integral membrane protein has an intracellular domain larger than Mr 12,000; it does not have an extracellular one. One of the monoclonal antibodies was employed for the isolation of band 7 integral membrane protein by immunoaffinity chromatography; subsequent Edman degradation revealed a blocked N-terminus.     

The relation between the soluble factor associated with H(+)-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli.

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H(+)-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Ths) and a membrane-bound component are together required for activity. Ths was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an Mr of approx. 74,000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, Mr 43,000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Ths to restore activity to depleted membranes. They reacted with a polypeptide of Mr 43,000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43,000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha subunit of the E. coli protein. The break between the alpha and beta polypeptides of E. coli transhydrogenase is such that both components are membrane-associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Ths has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Ths and the reported sequence of alanine dehydrogenase from Bacillus but Ths did not have any alanine dehydrogenase activity.     

A new trophoblast-derived growth factor from human placenta: purification and receptor identification

This paper describes the identification and characterization of a new peptide growth factor. The peptide was isolated from trophoblastic brush border membranes of human placenta. The purified preparation was homogeneous and consisted of a single polypeptide of Mr 34 000 with a pI of about 6.0. This peptide stimulated DNA replication in cultured fibroblasts. The following association was seen between activity and protein: During DEAE-cellulose chromatography, both the 34-kilodalton (kDa) protein and the mitogenic activity displayed identical binding and salt dependence of elution. Nondenaturing electrophoresis at pH 8.3 revealed a comigration of the 34-kDa protein and the DNA replication stimulatory activity. Identical electrophoretic mobilities were displayed for both activity and protein at pH 7.0. These results demonstrate that the preparation is homogeneous and show that growth factor activity is intrinsic to the 34-kDa polypeptide. Binding of the 125I-labeled 34-kDa mitogen to target fibroblastic cells was specific; i.e., nanomolar concentrations of the unlabeled 34-kDa protein competed effectively with the labeled protein, whereas a variety of well-characterized growth factors and hormones were unable to compete even at micromolar levels. Thus the 34-kDa protein interacts with target cells through highly specific surface receptors. Chemical cross-linking techniques were used to investigate the identity of the receptor for the 34-kDa mitogen. Cross-linking of fibroblastic cells containing bound 125I-labeled 34-kDa protein generated a radiolabeled complex of 86 kDa in all four cell types examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 23-kDa polypeptide from Common Frog lens: isolation,characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.     

A possible ligand of serum origin for the kidney autoantigen of Heymann nephritis

Earlier studies have localized the Heymann nephritis (HN) autoantigen (gp330) in the coated pits of the plasma membrane and multivesicular bodies of the glomerular epithelial cell. Because of these locations in the glomerular epithelial cells, it has been suggested that the HN Ag may be a receptor. The aim of our study was to search for a ligand which can bind the HN autoantigen. Normal rat serum was subjected to SDS-PAGE under reducing and non-reducing conditions followed by Western analysis of the separated polypeptides. A reaction was revealed directly by autoradiography using 125I labeled HN autoantigen as a probe and indirectly by enzyme immunodetection using unlabeled nephritogenic autoantibody (anti-gp330) eluted from glomeruli of diseased rats followed by biotinylated rabbit anti-rat IgG avidin-peroxidase complex. A polypeptide of 76 kDa Mr was identified under non-reducing conditions as a serum protein reacting with the HN autoantigen. Reactivity of the 76-kDa polypeptide was lost when serum was electrophoresed under reducing conditions. Direct binding of the 76-kDa polypeptide obtained from serum to the HN autoantigen obtained from kidney suggests that the 76-kDa polypeptide may be a ligand for the autoantigen. This is the first documentation of a possible ligand for the HN autoantigen. Not only does this polypeptide bind to the HN autoantigen but it also shows direct binding with the nephritogenic autoantibody eluted from glomerular deposits. This characteristic of the 76-kDa polypeptide indicates that this serum protein may potentially play a role in the development of the glomerular lesion of active HN. Further analysis of this serum component should assist in understanding the normal function of the HN autoantigen.     

Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D9K) is unstable when stored at 4 degrees C in the absence of Ca(II). Cleavage of the polypeptide occurs producing approximately 5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an Mr 13,700, in contrast to the observed Mr 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.     

Isolation and partial characterization of a 48-kDa protein which is induced in normal lymphocytes upon mitogenic stimulation

A 48-kDa protein (p48) crossreactive with an antiserum directed against the 12 C-terminal amino acids of the human cellular myc gene-encoded protein was isolated from a Burkitt lymphoma cell line. The p48 protein is a basic protein and has a cytoplasmic localization. An antiserum prepared against purified p48 reacts specifically with a 48-kDa protein present in a variety of mouse and human cells. This polypeptide is detected at very low levels in normal, resting, peripheral blood lymphocytes, but is induced several-fold by stimulation with either concanavalin A or pokeweed mitogen. The association of p48 induction with a proliferative response and the crossreactivity with the cellular myc protein are discussed.     

Identification of highly methylated arginine residues in an endogenous 20-kDa polypeptide in cancer cells.

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.     

The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity-purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48-kDa (alpha) and 58 kDa (beta) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93-kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane-spanning subunits of the GlyR and those of other ligand-gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of alpha subunits, and increased levels of the 93-kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned alpha subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion-conducting GlyR as a homolog of other ligand-gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.