Isolation and characterisation of monoclonal antibodies against hydrophobic membrane subunit 9 of the yeast mitochondrial H+-ATPase

Five stable lines of myeloma-spleen cell hybrids, producing antibodies against the proteolipid subunit 9 of the yeast mitochondrial H+-ATPase F0-sector, have been isolated by immunizing mice with a proteolipid preparation in the presence of sodium dodecyl sulphate. One of these monoclonal antibodies also reacted with subunit 8 of the enzyme complex indicating a shared epitope. The antibodies did not react with the holo-H+-ATPase, suggesting that their epitopes are shielded by other subunits of the enzyme complex.     

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Thermal stability of soluble mitochondrial H+-ATPase.

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the alpha-helix of H+-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20 degrees--40 degrees C. Above 40 degrees C, the pattern of thermal decomposition of the three-dimensional structure of H+-ATPase is independent of the nature of nucleotides present. Highly stable alpha-helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Fragmentation of mitochondrial H+-ATPase hydrophobic complex]

The subunits with molecular weights of 30 000, 10 000 and 20 000 + 19 000 have been obtained by fractionation of the hydrophobic part of the oligomycin-sensitive ATPase complex on Sephadex G-200 and Sephadex G-150 in the presence of sodium dodecyl sulfate.     

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+-ATPase (F1-F0), F0 of the H+-ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+-ATPase have been examined in the present paper. When ETPH and purified H+-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistently inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0, of H+-ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+-ATPase, showing competition for the FB dithiol group.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli

The H+-ATPase (F1F0) of Escherichia coli was purified from cells labeled with either [35S]sulfate or [U-14C-D] glucose, and the molar ratio of subunits in the complex determined. The molar ratio was calculated from the radioactivity incorporated into each subunit, using either the subunit sulfur content or subunit molecular weight. These labeling experiments confirm an alpha 3 beta 3 gamma 1 delta 1 epsilon 1 ratio of subunits in F1, and indicate a chi 1 psi 2 omega 10 ratio of subunits in F0. The chi, psi, and omega designations used here refer to the subunits of F0 in order of decreasing molecular weight. Staining with Coomassie brilliant blue gave a reliable indication of the molar ratio of subunits in F1, but very erroneous values for each of the subunits of F0. We attempted to estimate the ratio of subunits in the native membrane, since the stoichiometry determined for the purified complex could be an anomaly of purification. These estimates were made after labeling cells with [35S]sulfate during amplification of the ATPase genes carried on a lambda transducing phage. The subunit ratios in the native membrane were reasonably close to those obtained with purified F1F0. We conclude that the stoichiometry determined reflects the composition of F1F0 in the native membrane. The most surprising conclusion from this study is that there are 10 +/- 1 omega ("proteolipid") subunits in each F1F0 complex. This is considerably more than had been assumed previously.     

Monoclonal antibodies against tryptophanyl-tRNA-synthetase

Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme.     

Monoclonal antibodies against ovalbumin

Fusion of cells of the NS-1 mouse myeloma line with spleen cells from $\text{BALB}\text{c}$ mice immunized against ovalbumin produced hybrid cells which continuously secrete antibodies specific for ovalbumin. One of these cells was used to establish a cloned line. Studies of its antibody obtained either from ascites fluid or from medium from hybridoma cultures showed high titer and specificity against ovalbumin using the double antibody technique with rabbit anti-mouse immunobeads; the antibody proved to be of the IgG₁ (kappa) subclass and type.     

Monoclonal antibodies against calcitonin

Summary Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dotblot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde-or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu^1,7]-eel CT by bot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.Presented in part at the International Symposium on Biotechnology in Clinical Medicine, BIOTECH RIA '87, 13th–15th April, 1987, Rome, Italy     

Monoclonal antibodies against bacteria

Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

The long physiological reach of the yeast vacuolar H+-ATPase

V-ATPases are structurally conserved and functionally versatile proton pumps found in all eukaryotes. The yeast V-ATPase has emerged as a major model system, in part because yeast mutants lacking V-ATPase subunits (vma mutants) are viable and exhibit a distinctive Vma- phenotype. Yeast vma mutants are present in ordered collections of all non-essential yeast deletion mutants, and a number of additional phenotypes of these mutants have emerged in recent years from genomic screens. This review summarizes the many phenotypes that have been associated with vma mutants through genomic screening. The results suggest that V-ATPase activity is important for an unexpectedly wide range of cellular processes. For example, vma mutants are hypersensitive to multiple forms of oxidative stress, suggesting an antioxidant role for the V-ATPase. Consistent with such a role, vma mutants display oxidative protein damage and elevated levels of reactive oxygen species, even in the absence of an exogenous oxidant. This endogenous oxidative stress does not originate at the electron transport chain, and may be extra-mitochondrial, perhaps linked to defective metal ion homeostasis in the absence of a functional V-ATPase. Taken together, genomic data indicate that the physiological reach of the V-ATPase is much longer than anticipated. Further biochemical and genetic dissection is necessary to distinguish those physiological effects arising directly from the enzyme’s core functions in proton pumping and organelle acidification from those that reflect broader requirements for cellular pH homeostasis or alternative functions of V-ATPase subunits.     

Immunological cross-reactivity of fungal and yeast plasma membrane H+-ATPase

The plasma membrane H+-ATPases from fungi and yeasts have similar catalytic and molecular properties. A structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kDa polypeptide of the plasma membrane H+-ATPase from Neurospora crassa. A strong cross-reactivity is observed between the fungal H+-ATPase and the enzyme from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Structural homologies are indicated also by the analysis of the cross-reactive peptides originated by proteolytic digestion of Neurospora and S. cerevisiae purified enzymes. Neither enzyme from these two sources appears to be glycosylated by a highly sensitive concanavalin A affinity assay on blotted proteins. A glycoprotein of Mr 115000 and pI 4.8-5, which comigrates with a cell cycle-modulated protein on 2D gel, is present in partially purified preparations of plasma membrane H+-ATPase of S. cerevisiae and it is shown to be structurally unrelated to H+-ATPase.     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.