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C Carmeli

Publications and source records attributed to C Carmeli.

At least 19 recordsLinked to original sources
PubMed2000

Role of metal ions in coupling between chemical catalysis and conformational changes in ATP synthase.

Isolated chloroplast ATP synthase (CF0F1) was used for determination of the structure-function relation by measuring the effect of divalent metal ions on the properties of ATPase. Mg2+ ions were more efficient catalysts than Ca2+ ions as indicated by Kcat/Km of 55.2 and 5.4, respectively. Other activity parameters related to binding, such as the Km of MATP and Ki of MADP, indicated a stronger binding in the presence of Mg2+ as seen from a Mg2+/Ca2+ ratio of 2.8 and 3.8, respectively. Strong binding of Ca2+ ions with a Kd of 0.03 +/- 00.6 microM-1 was detected only in the presence of ADP probably because of the positive interactive effect of CaADP as indicated in the inhibition properties. Mg2+ ions were more efficient catalysts also in other forms of the enzyme such as in the thylakoid membrane, in isolated CF0F1 and in CF1. The Mg2+/Ca2+ ratio of Kcat/Km was 5.3, 10.2 and 1.5 for the thylakoid membrane enzyme, the isolated CF0F1 and the soluble CF1 respectively. This indicated that Ca2+ ions became less efficient catalysts in the more intact and integrated enzyme while Mg2+ ions were as efficient in all forms of the enzyme. Unlike Mg2+, Ca2+ ions also did not support proton-coupled ATP synthesis and ATP driven proton pumping. It is suggested that the differences in the ligand structure of these two ions might be the reason for the differential function. An average 0.3 A shorter bond length of octahedral first coordination in Ca2+ ions caused a weaker binding of CaATP than that of MgATP. The effect of differential binding is discussed in relation to the binding of the transition state intermediate and to the rate of product release.

Catalysis↗
PubMed1995

Epithelial-mesenchymal conversion of dermatome progenitors requires neural tube-derived signals: characterization of the role of Neurotrophin-3.

Development of the somite-derived dermatome involves conversion of the epithelial dermatome progenitors into mesenchymal cells of the dermis. In chick embryos, neural tube-derived signals are required for this conversion, as the interposition of a membrane between neural tube and somites results in a failure of the dermatome to lose its epithelial arrangement. However, dermis formation can be completely rescued by coating the membranes with Neurotrophin-3, but not with the related molecule Nerve growth factor. Neurotrophin-3 was also found to be necessary for dermatome dissociation using in vitro explants or partially dissociated dermomyotomes. The functional relevance of these observations was investigated by neutralizing endogenous Neurotrophin-3 using a specific blocking antibody. Antibody-treated embryos revealed the presence of tightly aggregated cells between myotome and ectoderm instead of the loose dermal mesenchyme observed in embryos treated with control antibodies. As previous studies have demonstrated the presence of Neurotrophin-3 in the neural tube, these results suggest that it may be a necessary neural tube-derived signal required for early stages of dermis formation.

Animals↗
PubMed1993

Vanadate, a transition state inhibitor of chloroplast CF1-ATPase.

The activity of CF1-ATPase was inhibited by vanadate in an allosteric manner with respect to CaATP as substrate. The cooperative interaction was enhanced by preincubation of the enzyme in the presence of ADP and Ca2+ ions and of free divalent metal ions during assay of the activity. The strongest cooperative interaction with a Hill coefficient of 5.3 +/- 0.1 was found when the reaction was stopped after 30 s, before steady state was reached. Under these conditions, the concentration of an exchangeable ADP, tightly bound to one of the active sites on the enzyme, was shown to be the highest. A Ki of 12.4 +/- 1.2 microM for vanadate inhibition was determined under these conditions. Direct measurements with the aid of 51V NMR indicated that vanadate binds to CF1 in the presence of Ca2+ and ADP in a positive cooperative manner with a Hill coefficient of 2.3 +/- 0.2 and an average Kd of 0.3 +/- 0.04 nM. It was suggested that a formation of pentacovalent vanadyl-ADP at the active site caused the inhibition. Vanadyl-ADP was suggested to be a strong inhibitor, being an analogue of a pentacovalent phosphoryl-ADP, which is proposed to be the transition state intermediate of CF1.

Adenosine Diphosphate↗
PubMed1993

Neurotrophin-3 affects proliferation and differentiation of distinct neural crest cells and is present in the early neural tube of avian embryos.

Neurotrophin-3 is mitogenic for cultured quail neural crest cells (Kalcheim et al., 1992, Proc. Natl. Acad. Sci. USA 89:1661-1665). We now report that neurotrophin-3 also influences the survival and/or differentiation of a subset of postmitotic neural crest precursors into neurons, provided these progenitors are grown on a cellular substrate. When cultured for 1 day on monolayers of NT-3-producing, chinese hamster ovary cells, 59% of the neural crest clusters growing on the transfected line revealed the presence of intense neuronal outgrowth, compared to 25% of that in controls. Moreover, dissociated neural crest cells grown for 20 h on top of mesodermal cells in the presence of various concentrations of purified recombinant neurotrophin-3 displayed a dose-dependent increase in neuronal number. Localization experiments using specific polyclonal antibodies, revealed that neurotrophin-3 is confined to neuroepithelial cells of quail neural tubes in situ on E2 and E3, and to E2 neural tubes grown in culture for 24 h. At this stage, neural crest cells and somites were negative. At later stages, staining was likewise apparent in peripheral nerves and dorsal root ganglia. We, therefore, propose that NT-3, a factor that is expressed in the early avian central nervous system, has multiple effects both on the proliferation and differentiation of distinct neural crest cells, which depend on the state of commitment of the responsive progenitors.

Animals↗
PubMed1992

Inhibition of chloroplast CF1-ATPase by vanadate.

Inhibition of ATPase activity by vanadate, having K1/2 of 0.5 mM, was demonstrated in the CF1-ATPase. The Ca(2+)-dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 +/- 0.6. Vanadate also inhibited ATPase and Pi-ATP exchange activities of the chloroplast membrane-bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1-ATPase is discussed.

Adenosine Diphosphate↗
PubMed1992

Neurotrophin 3 is a mitogen for cultured neural crest cells.

Neurotrophin 3 (NT-3) promotes the survival and induces neurite outgrowth from a subset of neural crest (NC) and placode-derived neurons. We now report that this growth factor regulates the proliferation of cultured NC progenitor cells grown in a serum-free defined medium. In cultures of somites containing NC cells at migratory stages, NT-3 promotes a 2- to 8.4-fold increase in the number of NC cells incorporating [3H]thymidine into nuclei and a 1.8- to 4.8-fold increase in NC cell number compared to controls without added factor. NT-3 also promoted, to a lesser extent, the proliferation of NC cells in homogeneous cultures established from NC clusters. In addition to its effect on NC cells, NT-3 was mitogenic to somite cells in the mixed NC/somite cultures. These data demonstrate that NT-3 can act directly on the NC cells. They also indicate that the response of NC cells to NT-3 may be modulated by the presence of somitic cells. We suggest that NT-3 may be one of the central nervous system-derived factors that mediate NC cell proliferation in vivo.

Animals↗
PubMed1991

Spheroplast-derived membrane vesicles from Rhodobacter capsulatus cells catalyzing nucleotide transport.

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.

Adenosine Diphosphate↗
PubMed1990

Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase.

The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.

Binding Sites↗
PubMed1989

Nucleotide transport in Rhodobacter capsulatus.

Cytoplasmic membrane vesicles isolated from the gram-negative photosynthetic bacterium Rhodobacter capsulatus catalyzed the transport of nucleotides. No transport occurred in the intact bacteria unless they were pretreated with EDTA. The transport rate was measured by incorporation of radioactive phosphate into externally added ADP or by incorporation of nonradioactive phosphate into added labeled ADP. The catalytic activities which utilized the added ADP were photosynthetic ATP synthesis, Pi-ADP exchange, and adenylate kinase. These activities were shown to occur on the cytoplasmic side of the internal membrane. The products were found in the outer medium. The rate of nucleotide transport across the membranes was comparable to the rate of photophosphorylation. These results indicated that nucleotides can be transported across the cytoplasmic membrane but not across the outer membrane of the native R. capsulatus cell. Therefore, by analogy to the mitochondrial ATP-ADP translocator, the exchange might function as an energy transfer system to the periplasm of these bacteria.

Adenine Nucleotides↗
PubMed1986

Extended X-ray absorption fine structure of Mn2+ and Mn2+ X ATP complex bound to coupling factor 1 of the H+-ATPase from chloroplasts.

The spinach chloroplast ATPase, coupling factor 1, contains three tight Mn2+-binding sites which interact cooperatively. The bound manganese coordinations were studied by x-ray absorption fine structure analysis. Mn2+ was found to be bound to the enzyme with an average Mn-O bond length of 2.15 +/- 0.15 A, significantly shorter than the 2.15 +/- 0.15 A of the Mn-O bond of the average first hydration shell for Mn2+ in aqueous solution. On adding ATP to the manganese-enzyme mixture, a tertiary complex of Mn2+ X ATP X enzyme was formed as indicated by the appearance of a second shell. Mn-P bond distances were estimated at 4.95 +/- 0.15 A in the tertiary Mn2+ X ATP X enzyme complex, which was considerably longer than the Mn-P bond distance of 3.36 +/- 0.15 A for the Mn2+ X ATP complex in aqueous solution. The Mn-P bond distance in the tertiary Mn2+ X ATP X enzyme complex decreased to 4.32 +/- 0.15 A when selenite, a potent effector of ATPase activity, was added. Based on these results, it is suggested that the tertiary complex is required for catalysis. The stimulation of ATP hydrolysis by anions such as selenite may be the result of shortening the distance between Mn2+ and the ATP phosphates in the enzyme active site.

Adenosine Triphosphate↗
PubMed1985

Cooperativity among manganese-binding sites in the H+-ATPase of chloroplasts.

Coupling factor, isolated from lettuce chloroplasts, contained several binding sites for Mn2+ ions. Three of these sites showed strong cooperative interactions having a Hill coefficient of 2.9 +/- 0.20 and a Kd of 14.7 +/- 0.44 microM. Three additional non-interacting Mn2+-binding sites were found with a Kd of 46.7 +/- 2.3 microM. Chemical modification with naphthylglyoxal of 1 arginyl residue/chloroplast coupling factor 1, which inhibited ATPase activity, inhibited the cooperativity among the sites but did not prevent Mn2+ binding to the enzyme. It is suggested that the cooperative interaction among the Mn2+-binding sites is an expression of the interaction among the active sites of the enzyme which is required for catalysis.

Adenosine Triphosphate↗
PubMed1981

Modulation by bicarbonate, phosphate, and maleate of the kinetics of adenosinetriphosphatase activity and of the binding of manganese ions to chloroplast coupling factor 1.

Bicarbonate, maleate, and phosphate were shown to modulate adenosinetriphosphatase (ATPase) activity in coupling factor 1 from chloroplasts. Kinetic analysis of the changes in the ratio between the apparent Km with and without effectors indicated that the stimulation of the activity by bicarbonate was a result of a decrease in the Km for MnATP2-. The inhibition by phosphate resulted from a decrease in the Ki for free ATP as a competitive inhibitor at pH 8. THe effectors did not change Vmax at this pH. However, at pH 6.5, both Km and Vmax of ATPase activity with MnATP2- were changed by maleate, yet the mode of inhibition by free ATP remained unaltered. In addition to decreasing the Km, bicarbonate induced a 10-fold decrease in the Kd for binding of Mn2+ at the two tight binding sites in the presence of ATP at pH 8. At pH 6.5, maleate also decreased both the Km for MnATP2- and the Kd for Mn2+ binding. A decrease in the Km of a substrate induced by an effector is likely to be a result of a decrease in the binding constant of the substrate. Therefore, these results are in harmony with the suggested assignment of the two tight binding sites of Mn2+ at the active sites of the enzyme.

Adenosine Diphosphate↗
PubMed1981

Modulation by divalent metal ions of the autocatalytic reactivity of adenosinetriphosphatase from chloroplasts.

A nonlinear, pre-steady-state initial rate of ATP hydrolysis is obtained on the addition of a divalent metal ion--ATP complex to a heat-activated coupling factor 1 isolated from chloroplasts. The acceleration of the initial rate follows first-order kinetics. The observed first-order kinetic constant (kobsd) changes with the concentration of the substrate, reaching half-maximal value at the Km for ATP hydrolysis. Preincubation of the enzyme with divalent metal ions decreases the kobsd from 1 to 0.04 s-1. Saturation of the divalent metal ion effect was obtained at the micromolar range. It is suggested that the autocatalysis is a result of early stages in ATP hydrolysis which induce conformational changes in the enzyme. Binding of divalent metal ions in the absence of ATP slows down this change.

Adenosine Triphosphatases↗