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PubMed1991

Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled [18O]Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium [32P]ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix. The rate of bound ATP formation from bound Pi at catalytic sites was over twice the rate of the overall reaction in both states 4 and 3. The rate of reaction at the catalytic site is considerably less sensitive to the decrease in membrane potential and the concentration of medium ADP than is the rate of medium ATP formation. This supports the view that the active catalytic site is occluded and proceeds at a rapid rate which is relatively independent of delta psi m and of media substrates.

Adenosine Triphosphate↗
PubMed1991

Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.

Adenine Nucleotides↗
PubMed1991

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.

Adenosine Triphosphate↗
PubMed1991

Two genes, atpC1 and atpC2, for the gamma subunit of Arabidopsis thaliana chloroplast ATP synthase.

Arabidopsis thaliana has two genes (atpC1, atpC2) coding for gamma subunits of chloroplast ATP synthase. The atpC1 and atpC2 were cloned and sequenced. They had no introns within the reading frames and coded for proteins of 373 and 386 amino acid residues, respectively, including putative transit sequences (50 and 60 amino acid residues, respectively). In contrast, the spinach gamma subunit gene had two introns within the reading frame. The mature sequences coded by the two genes of A. thaliana (atpC1, 323 residues; atpC2, 326 residues) were homologous with that of spinach (J. Miki, M. Maeda, Y. Mukohata, and M. Futai (1988) FEBS Lett. 232, 221-226): the homologies of gamma subunits coded by atpC1 and atpC2 were 72%, those of the subunits coded by atpC1 and spinach cDNA were 84%, and those of the proteins coded by atpC2 and spinach cDNA were 71%. Like the spinach subunit, the gamma subunits coded by the two genes had unique regulatory domains not found in mitochondrial or bacterial subunits. Poly(A)+ mRNAs corresponding to atpC1 (1.5 kilobases) and atpC2 (2.5 kilobases) were detected in illuminated plants, the amount of the former being at least 140 times that of the latter. The atpC1 mRNA was not found in dark-adapted plants. Nuclear protein(s) specifically bound to the upstream region of atpC1 was detected by gel shift assay and its binding was shown to be inhibited by the GT-1 element of the gene encoding the ribulose-1,5-bisphosphate carboxylase small subunit, which is expressed under illumination (P. J. Green, S. A. Kay, and N. H. Chau (1987) EMBO J. 6, 2543-2549). Consistent with these findings, an increased amount of the gamma subunit was detected immunochemically in illuminated plants.

Amino Acid Sequence↗
PubMed1991

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.

Adenosine Diphosphate↗
PubMed1991

Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase.

Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides. The alpha and beta subunits make up the bulk of protein mass of F1. In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment. Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described. Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria. In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S. cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized. The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587. The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein. In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein. The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion. Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane. The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer. This suggests that the ATP12 protein is probably part of a larger complex.

Amino Acid Sequence↗
PubMed1991

Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).

Adenosine Diphosphate↗
PubMed1978

[Stimulation mechanism of soluble ATPase from chloroplasts (CF1)].

Effects of various compounds on Mg-dependent ATPase activity of chloroplast coupling factor--CF1--were studied. It was shown that the stimulating effect of compounds is increased with the increase in their hydrophobicity. Under given experimental conditions all compounds under study readily accept and donate protons. The maximal efficiency is reached when pH of the medium is close to the pK value of conjugated acid. It is assumed that the stimulating effects of compounds on Mg-dependent chloroplast ATPase consist in the increase of the rate of the limiting step of enzyme induced proton translocation coupled to the catalytic step of ATP hydrolysis.

2,2'-Dipyridyl↗
PubMed1992

Two unrelated alkaliphilic Bacillus species possess identical deviations in sequence from those of other prokaryotes in regions of F0 proposed to be involved in proton translocation through the ATP synthase.

The a and c subunits of two unrelated alkaliphilic Bacillus species contain unusual motifs in regions previously implicated by others in H(+)-coupled oxidative phosphorylation. The facultative alkaliphile B. firmus OF4 apparently does not contain genes encoding an alternative F0, supporting other evidence that a single species of proton-translocating F1F0-ATPase catalyses oxidative phosphorylation both at low and high pH. The unusual F0 sequence motifs may be part of the adaptation of the extreme alkaliphiles to growth at very high pH.

Amino Acid Sequence↗
PubMed1992

pH regulation by Streptococcus mutans.

The intracellular pH (pHi) optimum for glycolysis in Streptococcus mutans Ingbritt was determined to be 7.0 by use of the ionophore gramicidin for manipulation of pHi. Glycolytic activity decreased to zero as the pHi was lowered from 7.0 to 5.0. In contrast, glycolysis had an extracellular pH (pHo) optimum of 6.0 with a much broader profile. The relative insensitivity of glycolysis to the lowering of pHo was attributed to the ability of S. mutans to maintain a transmembrane pH gradient (delta pH, inside more alkaline) at low pHo. At a pHo of 5.0, glycolyzing cells of S. mutans maintained a delta pH of 1.37 +/- 0.09 units. The maintenance of this delta pH was dependent on the concentration of potassium ions in the extracellular medium. Potassium was rapidly taken up by glycolyzing cells of S. mutans at a rate of 70 nmol/mg dry weight/min. This uptake was dependent on the presence of both ATP and a proton motive-force (delta p). The addition of N-N'-dicyclohexylcarbodiimide (DCCD) to glycolyzing cells of S. mutans caused a partial collapse of the delta pH. Growth of S. mutants at pHo 5.5 in continuous culture resulted in the maintenance of a delta pH larger than that produced by cells grown at pH 7.0. These results suggest the presence of a proton-translocating F1Fo-ATPase in S. mutans whose activity is regulated by the intracellular pH and transmembrane electrical potential (delta psi). The production of an artificial delta p of 124 mV across the cell membrane of S. mutans did not result in proton movement through the F1Fo-ATPase coupled to ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed1976

Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3. The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.

Adenosine Triphosphatases↗
PubMed1975

Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3' :5'-monophosphate in the yeast Schizosaccharomyces pombe.

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h- wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cerevisiae strain IL 216-IA. The mediated uptake of 2-D-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport is not necessarily of mitochondrial origin. The uptake of 2-D-deoxyglucose is unaffected by the respiratory inhibitors. Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-'monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake. In the absence of respiration, Dio-9, and ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport. In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venruricidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate. These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-9-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.

Adenosine Diphosphate↗
PubMed1978

Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex.

A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.

Adenosine Triphosphatases↗
PubMed1978

The properties of adenosine triphosphatase from exponential and synchronous cultures of Alcaligenes eutrophus H16.

The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (K(i) 142mum) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; K(i) 750mum) by NN'-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN'-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but -->H(+)/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules.

Affinity Labels↗
PubMed1979

The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity.

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.

Adenosine Triphosphatases↗
PubMed1979

The ATP synthetase of Escherichia coli K12: purification of the enzyme and reconstitution of energy-transducing activities.

The ATP synthetase of Escherichia coli K12 was purified by a simple procedure. The dicyclohexylcarbodiimide-sensitive ATPase activity was enriched 21-fold. The ATP synthetase preparation contained the eight polypeptides (alpha, beta, gamma, a,delta, b,espilon, c) of the enzyme and a residual contamination (4% of the total protein) as shown by dodecylsulfate/polyacrylamide electrophoresis. The polypeptide c was specifically labelled with [14C]dicyclohexylcarbodiimide. Energy-transducing activities were reconstituted from soybean phospholipids and the purified enzyme. The proteoliposomes exhibited a significantly higher ATP-32Pi exchange activity and a higher proton-translocating activity as compared to the untreated membranes.

Adenosine Diphosphate↗