Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris

A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2+, and the (Mg-PPi) complex is regarded to be the functional substrate. Free Mg2+ revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2+. Mg2+ is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2+ revealed a non-competitive type of inhibition on the Mg2+-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2+, had to be applied. An enrichment method along with stepwise lowering the concentrations of Triton X-100 and Mg2+ after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2+ was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity.     

Purification and properties of a soluble inorganic pyrophosphatase from Rhodopseudomonas palustris

A soluble inorganic pyrophosphatase from photolithoautotrophically grown Rhodopseudomonas palustris was purified to a state of apparent homogeneity applying high resolving liquid chromatography steps. Values of 65 500 and 64 500 were calculated for the relative molecular mass under non-dissociating conditions employing gel filtration and high-performance liquid chromatography, respectively. Dissociation sodium dodecyl sulfate gel electrophoresis resulted in a value of 32 000, indicating that the enzyme is composed of two subunits of equal molecular mass. Isoelectric focusing revealed a pI value of 4.7. The purified enzyme was specific for PPi and the activity was modified by divalent cations. Ca2+, Mn2+, Mg2+ and Co2+ were potent activators at a concentration ratio of [Me2+]/[PPi] less than 1. Ca2+ turned out to be the most potent activator. Free Me2+ was inhibitory on the PPiase activity. The (Me-PPi) complex is regarded as the functional substrate. Km and Ki values of the metal activation and inhibition were determined. An activation energy of Ea = 14.4 kJ/mol was derived from Arrhenius plots for the enzymatic reaction.     

Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris

A proteomics approach was evaluated for analysis of photosyntheis-related proteins that are characteristic of chromatophores, particles derived from purple phototrophic bacterial intracytoplasmic membranes. Proteins of purified chromatophores from Rhodopseudomonas palustris were solubilized and digested with trypsin, to create a collection of peptides that were fractionated by liquid chromatography. Peptide sequences were determined and assigned to specific proteins by analysis of tandem mass spectra of peptides, and comparison to a library derived from the recently determined R. palustris genome sequence. A total of 300 proteins were detected with a probability value >/=0.9, and the number of proteins detected increased to 345 when the minimum probability value was reduced to 0.5. Membrane-integral proteins of the reaction center, cytochrome b/c (1), light-harvesting and ATPase complexes were used as controls to assess how well this approach performs with hydrophobic proteins. New genes were identified, and tentatively designated as encoding photosynthesis-related proteins. We conclude that this approach is a powerful method to evaluate the possible existence of new photosynthesis-related proteins (and genes), although alternative methods are needed to evaluate the exact functions of newly discovered genes.     

Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase

An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized. ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite. The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes. Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases. ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase. N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases. Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.     

Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

A novel ATPase was solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, with low ionic strength buffer containing EDTA. The enzyme was purified to homogeneity by hydrophobic chromatography and gel filtration. The molecular weight of the purified enzyme was estimated to be 360,000. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate revealed that it consisted of three kinds of subunits, alpha, beta, and gamma, whose molecular weights were approximately 69,000, 54,000, and 28,000, respectively, and the most probable subunit stoichiometry was alpha 3 beta 3 gamma 1. The purified ATPase hydrolyzed ATP, GTP, ITP, and CTP but not UTP, ADP, AMP, or p-nitrophenylphosphate. The enzyme was highly heat stable and showed an optimal temperature of 85 degrees C. It showed an optimal pH of around 5, very little activity at neutral pH, and another small activity peak at pH 8.5. The ATPase activity was significantly stimulated by bisulfite and bicarbonate ions, the optimal pH remaining unchanged. The Lineweaver-Burk plot was linear, and the Km for ATP and the Vmax were estimated to be 1.6 mM and 13 mumol Pi.mg.-1.min-1, respectively, at pH 5.2 at 60 degrees C in the presence of bisulfite. The chemical modification reagent, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, caused inactivation of the ATPase activity although the enzyme was not inhibited by N,N'-dicyclohexylcarbodiimide, N-ethyl-maleimide, azide or vanadate. These results suggest that the ATPase purified from membranes of S. acidocaldarius resembles other archaebacterial ATPases, although a counterpart of the gamma subunit has not been found in the latter. The relationship of the S. acidocaldarius ATPase to other ion-transporting ATPases, such as F0F1 type or E1E2 type ATPases, was discussed.     

Major ATPases on clofibrate-induced rat liver peroxisomes are not associated with 70 kDa peroxisomal membrane protein (PMP70).

We previously reported that novel Mg(2+)-ATPases were induced in rat liver peroxisomes by clofibrate administration and that these activities consisted of at least two types of enzymes, N-ethylmaleimide (NEM)-sensitive and -resistant. Here we present evidence that neither of these major peroxisomal ATPases is associated with the 70-kDa peroxisomal membrane protein (PMP70), because: (i) proteinase K treatment of peroxisomes resulted in inactivation of only NEM-sensitive ATPase, whereas disappeared PMP70 completely; (ii) NEM-sensitive ATPase activity was barely immunoprecipitated with anti-PMP70 IgG; (iii) the solubilized ATPases behaved differently from PMP70 on native PAGE; and finally (iv), the major peroxisomal ATPases were separated from PMP70 on gel filtration chromatography.     

A vacuolar-type ATPase, partially purified from potassium transporting plasma membranes of tobacco hornworm midgut

An azide- and vanadate-insensitive, N-ethylmaleimide-sensitive ATPase has been partially purified from a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. The properties of the membrane-bound ATPase activity were identical to those of the ATPase activity of highly purified goblet cell apical membranes (Wieczorek, H., Wolfersberger, M. G., Cioffi, M., and Harvey, W. R. (1986) Biochim. Biophys. Acta 857, 271-281). 90% of the azide- and vanadate-insensitive ATPase activity was solubilized by C12E10, leaving 90% of the contaminating azide-sensitive mitochondrial ATPase activity in the pellet after centrifugation at 100,000 x g for 1 h. After discontinuous sucrose gradient centrifugation of the supernatant at 220,000 x g for 1 h nearly all of the azide- and vanadate-insensitive ATPase activity was found in the 30% sucrose fraction without contaminating azide- or vanadate-sensitive ATPase activity. Two prominent bands with relative molecular masses (Mr) of about 600,000 and 900,000, both displaying azide-insensitive and N-ethylmaleimide-sensitive ATPase activity, were found in native microgradient polyacrylamide gel electrophoresis of the 30% sucrose fraction. The two bands could not be separated by anion exchange chromatography. Denaturation of both bands resulted in the same polypeptide pattern (five major bands with Mr 70,000, 57,000, 46,000, 29,000 and 17,000) in sodium dodecylsulfate-polyacrylamide gel electrophoresis, indicating that they represented oligomers of the same protein unit. Substrate and inhibitor specificities of the partially purified ATPase were similar to those of the membrane-bound ATPase activity, whereas salt selectivity differed partly. Altogether, structural and functional properties of the ATPase strongly resemble those of vacuolar-type ATPases.     

The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

A membrane-bound ATPase from Halobacterium saccharovorum was solubilized using sodium deoxycholate and Zwittergent 3-10 and purified by hydrophobic and ammonium sulfate-mediated chromatography. The enzyme, which had a molecular mass of 350 kDa, was composed of two major (87 and 60 kDa) and two minor (29 kDa and 20 kDa) subunits. The halobacterial ATPases appear to be unlike any other ATPase described to date.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and characterization of an oligomycin and N,N'-dicyclohexylcarbodiimide-sensitive (Ca+ + Mg2+)-ATPase.

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin has been solubilized from Rhodospirillum rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified RrFo . F1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-sensitive, water-soluble RrF1 ATPase. The ATPase activity of RrF0 . F1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and from detailed kinetic studies it is concluded that the divalent cation-ATP complex is the substrate for both ATPase complexes. Free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 muM, respectively. The subunit composition of the purified RrFo . F1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggests that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.     

The proteolipid of the A(1)A(0) ATP synthase from Methanococcus jannaschii has six predicted transmembrane helices but only two proton-translocating carboxyl groups.

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general.     

The F1-type ATPase in anaerobic Lactobacillus casei

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.     

Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum

Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.     

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.     

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.     

Purification and properties of an acetate kinase from Rhodopseudomonas palustris

An acetate kinase from the photolithoautotrophically grown purple bacterium Rhodopseudomonas palustris was purified to apparent homogeneity by use of high resolving liquid chromatography steps. The monomeric enzyme was characterized by a relative molecular mass of 46,500 and an isoelectric point of 4.9. There was an absolute requirement for divalent metal ions in the enzymatic reaction. Mg2+ and Mn2+ were the most activating cations. The acetate kinase used pyrimidine and purine nucleotides almost equally well as phosphoryl donors. The enzyme phosphorylated acetate, propionate, butyrate and isobutyrate. ATP and acetate revealed the lowest apparent Km values and seemed to act as the favoured substrates. The apparent Km values for ATP formation were considerable lower than those for the formation of acetyl phosphate. The activation energy Ea = 21 kJ/mol of the acetyl phosphate formation was determined by application of Arrhenius plots.     

Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.     

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.     

The plasma membrane ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Purification and immunological relationships to F1-ATPases

The plasma-membrane-associated ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius characterized in a previous work [M. Lübben & G. Sch?fer (1987) Eur. J. Biochem. 164, 533-540] has been solubilized. It can be easily removed from the membrane by mild treatment with zwitterionic detergents, therefore it appears to be a peripheral membrane protein analogous to the soluble F1-ATPase of eubacteria and eukaryotes. Further purification has been achieved by subsequent gel permeation and ion-exchange chromatography. The final purity is greater than 70% as judged by staining intensities after SDS/polyacrylamide gel electrophoresis. The ATPase consists of two major polypeptides of 65 kDa (alpha) and 51 kDa (beta) in comparable quantities; a minor band (20 kDa) is assumed to be a contaminant or a constitutive part of the enzyme, possibly copurified in substoichiometric amount. The native molecular mass of the solubilized ATPase determined by gel permeation is 430 kDa. Considering the precision of these methods, it remains open whether a 3:3 stoichiometry reflects the contribution of alpha and beta subunits to the quaternary structure, in analogy to known F1-ATPases. The catalytic properties resemble those of the membrane-bound state. There are two pH optima at 5.3 and 8.0 in the absence and only one optimum at 6.5 in the presence of the activating anion sulfite. Activity is strictly dependent on the divalent cations Mg2+ or Mn2+. ATP and dATP are hydrolyzed with highest rates; also other purine and pyrimidine nucleotides are cleaved significantly, but not ADP, pyrophosphate and p-nitrophenyl phosphate. The ATPase is insensitive to azide or vanadate but is inhibited by relatively low concentrations of nitrate. Polyclonal antisera have been raised against the beta subunit of the Sulfolobus ATPase. Cross-reactivities with cellular or membrane extracts of a number of archaebacteria, eubacteria and chloroplasts have been analyzed by means of Western blotting and immunodecoration. A strong cross-reactivity with other genera of the Sulfolobales is observed, also with Methanobacterium, Methanosarcina, Methanolobus and Halobacterium. Even membranes of the eubacterium Escherichia coli and of eukaryotic chloroplast react with the antibodies. With one exception, in all cases the molecular mass of the cross-reacting polypeptide falls in the range of 51-56 kDa. Only in Halobacterium halobium, bands at 66 and 68 kDa have been detected. In order to identify the cross-reacting polypeptides, the purified F1-ATPases of E. coli, chloroplasts and beef heart mitochondria have been tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ribosomal Proteins of Rhodopseudomonas palustris

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ribosomal proteins of Rhodopseudomonas palustris revealed that the 29S subunit lacked a high-molecular-weight protein. It is suggested that a high-molecular-weight protein may function in protecting ribosomal ribonucleic acid from ribonuclease degradation.