Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

A simple method to quantitatively measure polypeptide JHNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J coupling constant and the linewidth (_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J = 0.5(_1/2) – MW/5000 + 1.8 for TOCSY spectra and J = 0.6(_1/2) – MW/5000 – 0.9 for NOESY spectra, where _1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which _1/2 measurements can be made, means that J coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date.     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

Relationship between the octanol-water partition coefficient of tertiary amines and their effect of ‘selective’ uncoupling of photophosphorylation

A series of tertiary amines was investigated for effects on the transmembrane proton potential difference ( H), on photophosphorylation and on electron-flux control related to the intrathylakoid proton potential ( H_I), using isolated chloroplasts ofSpinacia oleracea L. As indicated by 9-aminoacridine fluorescence and [14C]methylamine uptake, all amines studied inhibited a build-up of H and, in parallel, ATP synthesis. Even when H was low, strong H₁-dependent electron-flux control was observed under the influence of tertiary amines. The strength of flux control in the presence of low H and the effectiveness of inhibition of ATP synthesis linearly increased with the lipophilicity of the amines. The most effective of the amines tested caused 50% inhibition of ATP synthesis at a concentration of 6 M, which is about 1000-fold lower than the concentration required for inhibition by methylamine. The data presented indicate the existence of two proton domains in the thylakoid vesicles, one of them feeding the ATP-synthase, the other the sites of pH-dependent electron-flux control. It is concluded that tertiary amines develop their action in a lipophilic domain of the thylakoid membrane, in the vicinity of the ATP-synthase complex. A mechanism for selective uncoupling and for the maintenance of H_I-dependent electron flux control in the presence of low H is discussed.Abbreviations and symbols coefficient for pH-dependent electron flux control - 9-AA 9-aminoacridine - Chl chlorophyll - I₅₀ amine concentration producing 50% inhibition of ATP-synthesis - J_e flux of photosynthetic electron transport - k _H apparent rate constant for proton efflux - H₁ proton potential in the thylakoid lumen - H₁ transthylakoid proton potential difference - p partition coefficient - q _AA coefficient for 9-aminoacridine fluorescence quenching - PS photosystem - Q quantum flux of photosynthetically active light Dedicated to Professor Wilhelm Simonis, on the occasion of his 80th birthday     

The generation of the proton electrochemical potential and its role in energy transduction

Summary The evidence that all energy transducing membranes can generate a proton electrochemical potential difference, _H, across the membrane and that this potential can be used to transfer energy among energy transducing units and to generate ATP, has increased the interest for the view that _H plays an obligatory role in energy transduction and ATP synthesis. In the present article we shall concentrate on two experimental questions related with the generation and role of _H: (a) the charge/site ratio; (b) the relation between the proton electrochemical potential on one side and the cation electrochemical potential, the phosphate potential and the redox potential on the other. We shall then discuss the view that energy transduction corresponds to a molecular energy machine rather than to a fuel cell.     

The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

Characterization of Four Novel Variants of Goat βA-Globin Gene

Four novel alleles of the adult -globin gene of Capra hircus were observed in an extended study on hemoglobin polymorphism in goat breeds living in the island of Sardinia. Nucleotide sequencing showed that one of these alleles is due to a 2 bp substitution at codon 125 ( G, "LeuGlu). Two substitutions, the silent CT for Leu at codon 78 and the conservative A G (Lys Arg) at codon 104, are shared by the other three alleles, two of them having additional mutations, which suggests a common origin. The allele we provisionally called the ^Y shares four out of five amino acid substitutions, together with the same polymorphisms in the IVSII, we observed previously in the rather common ^E gene. This evidence allowed the origin of the ^E gene to be better characterized. The data increase to seven the number of alleles at the goat ^A -globin locus characterized thus far at the molecular level. A simplified nomenclature for the increasing number of goat -globin alleles is presented.     

The proton gradient across the vacuo-lysosomal membrane of lutoids from the latex ofHevea brasiliensis. I. Further evidence for a proton-translocating ATPase on the vacuo-lysosomal membrane of intact lutoids

Summary Lutoids (vacuo-lysosomal particles) were isolated from the latex ofHevea brasiliensis. Using flow dialysis with¹⁴C-methylamine uptake as a pH probe and⁸⁶Rb rubidium+valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a pH of 1 unit (interior more acid) and a of –70 mV (interior negative), when suspended in an isotonic medium at physiological concentration of potassium (30mm) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for pH in nonenergized lutoids. The addition of Mg-ATP (5mm) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons ( ) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of thein vivo maintenance of large lutoids/cytoplasm proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism.     

Modelling the dynamics of West Nile Virus

In this work we formulate and analyze a mathematical model for the transmission of West Nile Virus (WNV) infection between vector (mosquito) and avian population. We find the Basic Reproductive Number in terms of measurable epidemiological and demographic parameters. is the threshold condition that determines the dynamics of WNV infection: if the disease fades out, and for the disease remains endemic. Using experimental and field data we estimate for several species of birds. Numerical simulations of the temporal course of the infected bird proportion show damped oscillations approaching the endemic value.     

Energetics of CO formation and CO oxidation in cell suspensions of Acetobacterium woodii

Cell suspensions of Acetobacterium woodii produced CO from H₂ and CO₂. Depending on the conditions, more than 1,000 ppm CO were measured in the gas phase. This concentration was more than 10-fold higher than the thermodynamic equilibrium concentration that can be calculated to be 83.5 ppm for the experimental conditions used. This finding is taken as evidence that, besides the activation of formate, also CO production from CO₂ is an energy-dependent step in the reduction of CO₂ to acetate. Studies on the influence of ionophores and dicyclohexylcarbodiimide (DCCD) as well as that of CO and formaldehyde on acetate synthesis were undertaken in order to determine whether ATP or is the driving for CO₂ reduction to CO.Cells of A. woodii also catalyzed the conversion of CO (5% in the gas phase) to CO₂ and H₂. This process was coupled to the generation of metabolic energy, which could be used by the cells to drive the uptake of histidine into the cells; histidine uptake was almost completely inhibited by the ionophores valinomycin plus nigericin. The data were taken to indicate that in this acetogen the energy derived from CO oxidation can be converted to metabolic energy.Abbreviations DCCD dicyclohexylcarbodiimide - THF tetrahydrofolate - TCS tetrachlorosalicylanilide - TPP⁺ tetraphenylphosphonium ion - Val valinomycin; Nig, nigericin - DTT dithiothreitol - DTE dithioerythritol - DTE dithioerythritol - membrane potential - electrochemical proton potential - ppm parts per million     

Metabolic cost of sodium transport in toad urinary bladder

Summary The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential, , by continuous and simultaneous measurements of CO₂ production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO₂ and the passive permeability of the tissue. From these determinations active sodium transport,J _Na, and suprabasal CO₂ production, , were calculated. Since large transients inJ _Na and frequently accompanied any abrupt change in , steady state conditions were carefully defined.Some 20 to 40 min were required after a change in before steady state of transport activity and of CO₂ production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through the active transport pathway by an electrical gradient of –50 mV. In both cases the value of the ratio averaged some 20 sodium ions transported per molecule of CO₂ produced.When the Na pump was blocked by 10^–2 m ouabain, the perturbations of the transepithelial electrical potential did not elicit changes ofJ _Na nor, consequently, of .The independence of the ratio from over the range ±50 mV indicates a high degree of coupling between active sodium transport and metabolism.     

Effects of root temperature on uptake of nitrate and ammonium ions by barley grown in flowing-solution culture

Summary Barley plants (Hordeum vulgare L.) grown from seed for 28 days in flowing solution culture were subjected to different root temperatures (3, 5, 7, 9, 11, 13, 17, 25°C) for 14 days with a common air temperature of 25/15°C (day/night). Uptake of NH₄ and NO₃ ions was monitored separately and continuously from solutions maintained at 10 M NH₄NO₃ and pH 6.0. Effects of root temperature on unit absorption rate , flux and inflow were compared. After 5 days , and increased with temperature over the range 3–11°C for NH₄ ions and over the range 3–13°C for NO₃ ions, with little change for either ion above these temperatures. Q₁₀ temperature coefficients for NH₄ ions (3–13°C) were 1.9, 1.7 and 1.6 for , and respectively, the corresponding values for NO₃ ions being 5.0, 4.5 and 4.6. For both ions, , and changed with time as did their temperature dependence over the range 3–25°C, suggesting that rates of ontogenetic development and the extent of adaptation to temperature may have varied among treatments.     

Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Vanadium and molybdenum requirement for the fixation of molecular nitrogen by two Methanosarcina strains

Nitrogen fixation of the Methanosarcina barkeri strains Fusaro (DSM 804) and 227 (DSM 1538) was found to be dependent on the presence of vanadium or molybdenum whereby molybdenum (added as Na₂-molybdate) was preferred to vanadium (added as VCl₃). Strain 227 showed less pronounced effects on diazotrophic growth with respect to vanadium and molybdenum. Rhenium (ReCl₃) or tungsten (Na₂-tungstate) could not replace vanadium or molybdenum. The optimum concentrations were found to be 2M for vanadium and 5M for molybdenum (strain Fusaro). This Mo optimum of methanogenesis was 10-fold higher with N₂ than with NH₄Cl as nitrogen source. A vanadium requirement with NH₄Cl could not be detected. No interferences were observed if molybdenum and vanadium were added simultaneously under diazotrophic conditions. Growth yields were smallest for strain 227 grown diazotrophically ( =0.6g dw/mol in the presence of vanadium and =0.9g dw/mol in the presence of molybdenum), obviously higher for strain Fusaro grown diazotrophically ( =1.15g dw/mol in the presence of V and =1.4g dw/mol with Mo) and highest if M. barkeri was grown on NH₄Cl as N-source ( =3.4g dw/mol with Mo, strain Fusaro).     

Activity and oxygen consumption during hypoxic exposure in high altitude and lowland sceloporine lizards

Summary Resting rates of O₂ consumption against , exercise endurance times and during recovery from vigorous exercise were measured inSceloporus occidentalis captured near sea level and inS. graciosus captured above 2850 m. Oxygen consumption against was also measured inS. occidentalis captured above 2850 m. When was recorded continuously, as ambient was slowly reduced from 155 Torr, it became directly dependent upon ambient between 110 and 120 Torr. The critical for the high altitude lizards was lower than that for the lowland lizards, which enabled the former to maintain relatively higher 's when ambient was reduced below 120 Torr. The high altitude lizards also had significantly greater endurance when stimulated to exercise at 1600 m ( 130 Torr). Both the higher under hypoxia and the greater endurance roughly parallel a significantly greater maximum in the high altitude lizards. At a simulated altitude of 3600 m ( 100 Torr), maximum and rate of recovery of the O₂ debt calculated from post active were significantly reduced in the lowland but not the high altitude lizards. The effects of simulated altitude conditions on the lowland but not the mountaine animals indicate adaptations to altitude in these sceloporine lizards. We did not find any consistent relationship between organ/body weight ratios or hematocrit and our measures of endurance or the altitude at which the lizards were captured.     

Estimation of surface charges in some biological membranes

Summary The resting membrane potential data existing in the literature for the giant axon of the squid, frog muscle and barnacle muscle have been analyzed from the standpoint of the theory of membrane potential due to Kobatake and co-workers. The average values derived for the effective charge density (where is a constant, , and represents the fraction of counterions that are free, and is the stoichiometric charge density in the membrane) present on the different biomembranes existing in their normal ionic environment are 0.3, 0.325 and 0.17 M for the squid axon, frog and barnacle muscles, respectively. On the assumption that the values of are 0.4 and 0.2 for nerve and muscle membranes, respectively, values of 0.75, 1.62 and 0.85 M have been derived for the stoichiometric charge density present in the respective biological membranes. These correspond to 1 negative charge per 222, 103 and 195 Å of the membrane area of the squid axon, frog and barnacle muscles, respectively.     

Mathematical simulation of proton tracks in water vapor and their microdosimetric analysis

Summary The spatial pattern of primary physical events was calculated for protons in water vapor by means of a Monte Carlo program. Two different cross section data sets were used to cover the proton energy range from 0.2 to 15 MeV. From the spatial pattern of primary energy deposition, proximity functions were derived and from these the dose mean lineal energy was calculated. The contributions of different track components to for spherical target volumes of 1–100 nm were analysed. The results are compared with the LET approximation and with analytical calculations of based on expectation values of the radial energy deposition around the proton path (radial dose profiles). Finally the associated volume of proton tracks was calculated using the so called linear approximation, and energy deposition distributions were derived. These were compared with distributions calculated by means of restricted-LET.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Bioenergetics: the evolution of molecular mechanisms and the development of bioenergetic concepts

Possible routes for the evolution of cell energetics are considered. It is assumed that u.v. light was the primary energy source for the precursors of the primordial living cell and that primitive energetics might have been based on the use of the adenine moiety of ADP as the u.v. chromophore. It is proposed that the excitation of the adenine residue facilitated phosphorylation of its amino group with subsequent transfer of a phosphoryl group to the terminal phosphate of ADP to form ATP. ATP-driven carbohydrate synthesis is considered as a mechanism for storing u.v.-derived energy, which was then used in the dark. Glycolysis presumably produced compounds like ethanol and CO₂ which easily penetrate the membrane and therefore were lost by the cell. Later lactate-producing glycolysis appeared, the end product being non-penetrant and, hence, retained inside the cell to be utilized to regenerate carboxydrates when light energy became available. Production of lactate was accompanied by accumulation of equimolar H⁺. To avoid acidification of the cell interior, an F₀-type H⁺ channel was employed. Later it was supplemented with F₁. This allowed the ATP energy to be used for uphill H⁺ pumping to the medium, which was acidified due to glycolytic activity of the cells.In the subsequent course of evolution, u.v. light was replaced by visible light, which has lower energy but is less dangerous for the cell. It is assumed that bacteriorhodopsin, a simple and very stable light-driven H⁺ pump which still exists in halophilic and thermophilic Archaea, was the primary system utilizing visible light. The formed was used to reverse the H⁺-ATPase, which began to function as H⁺-ATP-synthase. Later, bacteriorhodopsin photosynthesis was substituted by a more efficient chlorophyll photosynthesis, producing not only ATP, but also carbohydrates. O₂, a side product of this process, was consumed by the H⁺-motive respiratory chain to form in the dark. At the next stage of evolution, a parallel energy-transducing mechanism appeared which employed Na⁺ instead of H⁺ as the coupling ion (the Na⁺ cycle). As a result, the bioenergetic system became more stable under unfavorable conditions. Apparently, the latest inventions of evolution of biological energy transducers are those which can utilize and outside the coupling membrane, like the bacterial flagellar motor and the TonB-mediated uphill transport of solutes across the outer membrane of bacteria.     

Linkage analysis using multiple DNA polymorphic markers in normal families and in families with fragile X syndrome

Summary Linkage data, using the polymorphic markers 52A (DXS51), F9, 4D-8(DXS98), and St14(DXS52), are presented from 14 fragile X pedigrees and from 7 normal pedigrees derived from the collection of the Centre d'Étude du Polymorphisme Humaine. A multipoint linkage analysis indicates that the most probable order of these four loci in normal families is DXS51-F9-DXS98-DXS52. Recombination frequencies ( ) corresponding to maximum LOD scores ( ) were obtained by two-point linkage analysis for a nuber of linkage groups, including: DXS51-F9 ( =5.94, =0.03), F9-DXS98 ( =0.51, =0.26), F9-DXS52 ( =0.84, =0.27), and DXS98-DXS52 ( =0.32, =0.20). A multipoint linkage analysis of these loci, including the fragile X locus, was also performed for the fragile X population and the data support the relative order (DSX51, F9, DXS98)-FRAXA-DXS52. Recombination frequencies and maximum LOD scores, which again were derived from two-point linkage analyses, were obtained for the linkage groups DXS51-F9 ( =9.96, =0) and F9-DXS52 ( =0.07, =0.45), as well as for the groups DXS51-FRAXA ( =2.42, =0.15), F9-FRAXA ( =1.30, =0.18), DXS98-FRAXA ( =0.05 =0.36), and DXS52-FRAXA ( =2.42 =0.15). The linkage data was further tested for the presence of genetic heterogeneity both within and between the fragile X and normal families for the intervals DXS51-F9, F9-DXS52, F9-FRAXA, and DXS52-FRAXA using a modification of the A test. Except for the interval F9-FRAXA (P<0.10) there was no evidence of genetic heterogeneity for each of the various linkage groups examined. The heterogeneity detected for the interval F9-FRAXA, however, was most likely due to one family (Fx-28) that displayed very tight linkage between these two loci.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential