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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The bound nucleotides of the beef-heart mitochondrial ATPase (F1) are lost during cold inactivation followed by (NH4)2SO4 precipitation. The release of tightly bound ATP parallels the loss of ATPase activity during this process. 2. During cold inactivation, the sedimentation coefficient (s20, w) of the ATPase first declines from 12.1 S to 9 S, then to 3.5 S. (NH4)2SO4 precipitation of the 9-S component also leads to dissociation into subunits with s20, w of 3.5 S. 3. The 9-S component still contains the bound nucleotides, which are removed when it dissociated into smaller subunits. 4. Reactivation of cold-inactivated ATPase by incubation at 30 degrees C is increased by the presence of 25% glycerol. ATP, however, does not have any clearcut effect on the degree of reactivation in the presence of glycerol. 5. ADP is an inhibitor of the reactivation, probably because it exchanges during reactivation for bound ATP giving rise to an inactive 12-S component. 6. The exchange of tightly bound nucleotides with added adenine nucleotides is more extensive and faster with cold-inactivated ATPase than with the native enzyme. During reactivation up to 1.6 moles of ATP and 1.0 mole ADP can exchange per mole enzyme. 7. Incubation with GTP, CTP or inorganic pyrophosphate induces an increased activity of the ATPase, which, however, soon declines in the presence of ATP. It also disappears on precipitation of GTP-treated enzyme with (NH4)2SO4.
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PMID:Nucleotide-binding properties of native and cold-treated mitochondrial ATPase. 12 64

The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry. The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites. The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites. Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven. For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects. Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons. In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.
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PMID:Calorimetric analysis of aspartate transcarbamylase from Escherichia coli: binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate. 23 71

Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter. Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no. 1, and B, which was 90% pure chromosome no. 2. The DNA of 10(6) chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E. coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/mumole). The cRNA product synthesized with template DNA from 10(5) sorted chromosomes contained more than 10(6) dpm. The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E. coli met-tRNAf. In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9 x 10(2) mole sec/l. Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l. This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E. coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis.
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PMID:Transcription and hybridization of 125I-cRNA from flow sorted chromosomes. 42 70

The interaction between the catalytic subunit (c3) and the regulatory subunit (r2) of aspartate transcarbamylase from Escherichia coli was studied by measuring the reversible formation of the c3r6 complex as a function of r2 concentration. Conversion to the native enzyme was prevented by using a very low concentration of c2 (40 ng per ml) in the presence of bovine serum albumin. A simple hyperbolic r2 saturation curve was obtained suggesting the presence of only one kind of c:r domain. From the association constant for the formation of c3r6, the free energy of c:r interaction can be estimated to be about -10 Cal per mole. Neither CTP nor ATP appears to affect the strength of c:r interaction in this complex. Succinate in the presence of carbamyl phosphate promotes tighter binding. At higher concentration of c3 and nonsaturating levels of r2, conversion to the native enzyme (c3r6) takes place. This renaturation process is second order with respect to the concentration of c3 and is virtually irreversible. Renaturation is inhibited by saturating levels of r2 and to some extent by both CTP and ATP. The effect of ligands on c:r interactions reported here may have significance in the allosteric mechanism of the native enzyme.
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PMID:Subunit interactions in aspartate transcarbamylase. The interaction between catalytic and regulatory subunits and the effect of ligands. 108 46

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle. 216 18

We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.
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PMID:Visualization and quantitative analysis of complex formation between E. coli RNA polymerase and an rRNA promoter in vitro. 305 11

1. The Ca movements in normal and ;ghost' L cells have been examined; all measurements were made using (45)Ca.2. Normal cells have a Ca concentration of about 1 m-mole/l. of cell volume, and exchange Ca in a complex way but with great rapidity; the time taken for the initial Ca(*) content to fall to half was less than 2 min.3. Poisoning normal cells with DNP 10(-3)M + IAA 10(-4)M causes a marked reduction in the Ca efflux, no change in Ca influx and an increase in total Ca.4. Variation in internal or external Na concentration does not alter the Ca fluxes or concentrations. Application of cyanide or ouabain and alteration of external K concentration had no effect on the Ca fluxes.5. The sulphydryl reagents, ethacrynic acid and N-ethylmaleimide (NEM), have a rapid and marked effect on reducing the Ca efflux.6. L cell ghosts previously poisoned with DNP+IAA have a low Ca efflux. When ATP or CTP is incorporated into such cells the Ca efflux becomes normal.7. An extra amount of phosphate is produced by L cell ghosts when pumping Ca. This is equivalent to the splitting of 1.8 moles of ATP per mole of Ca pumped.8. It is concluded that L cells have a Ca pump driven by ATP, and that Na has no effect on Ca movements in these cells.
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PMID:Effect of Na, metabolic inhibitors and ATP on Ca movements in L cells. 513 52

The Ca(++) transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca(++) from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of (45)Ca infused inside red cells indicated that over 95% of all Ca(++) in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP(1), CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca(++) transport does not appear to involve exchange of Ca(++) with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca(++) transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca(++) transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.
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PMID:Studies on the active transport of calcium in human red cells. 535 89

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.
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PMID:Substrate specificity of CTP synthetase from Escherichia coli. 675 17

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides and requires adenosylcobalamin (AdoCbl) as a cofactor. Recent cloning, sequencing, and expression of this protein [Booker, S., & Stubbe, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8352-8356] have now allowed its characterization by site-directed mutagenesis. The present study focuses on the role of five cysteines postulated to be required for catalysis. The choice of which of the ten cysteines of RTPR were to be mutated was based on extensive studies on the Escherichia coli ribonucleoside diphosphate reductase. Despite the differences between these two reductases in primary sequence, quaternary structure, and cofactor requirements, their mechanisms are strikingly similar. The mutagenesis studies reported herein further suggest that the complex role of the five cysteines is also very similar. A variety of single and double mutants of RTPR were prepared (C731S, C736S, C731 and 736S, C119S, C419S, C408S, and C305S), and their interaction with the normal substrate (CTP) was characterized under several sets of conditions. Mutants C731S, C736S, and C731 and 736S all catalyzed the formation of dCTP at rates similar to those of the wild-type (wt) enzyme in the presence of the artificial reductant DTT. In the presence of the in vivo reducing system (thioredoxin, thioredoxin reductase, and NADPH), however, each of these mutants catalyzed the formation of only 0.6-0.8 dCTPs per mole of enzyme. The inability of these mutants to catalyze multiple turnovers with respect to the in vivo reducing system suggests that their function might be to transfer reducing equivalents from thioredoxin into the active site disulfide of the reductase. Mutants C119S and C419S were targeted as being the active site cysteines, the ones which directly reduce the ribonucleotide substrate. As expected, neither of these mutants catalyzed the formation of dCTP. However, they did catalyze a time-dependent formation of cytosine, destruction of the cofactor, and the appearance of a chromophore associated with the protein--all phenotypes previously observed for the corresponding active site cysteines of the E. coli reductase. Mutant C408S was unable to catalyze dNTP production or cytosine release. Moreover, it was ineffective in catalyzing two additional reactions which are unique to this enzyme: the exchange of tritium from the 5' hydrogens of AdoCbl with H2O and the destruction of AdoCbl under anaerobic conditions to give 5'-deoxyadenosine and cob(II)alamin. These results are consistent with the role of this cysteine as the protein radical responsible for initiating catalysis.
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PMID:Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction. 791 94


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