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Query: UNIPROT:P06889 (Mol)
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Xenopus eggs contain large stores of glycogen, but this glycogen is not glycolytically processed during cleavage. The Embden-Meyerhof pathway is inhibited by the absence of pyruvate kinase activity in vivo, and lactate and pyruvate are present at relatively low levels. In the late blastula, just preceding gastrulation, lactate levels increase, indicating the onset of glycogen breakdown and glycolytic flux. Glycolysis from microinjected [14C]glucose-6-phosphate could be transiently activated, however, by the coinjection of ADP into fertilized eggs, and constitutively activated by the injection of the ATPase potato apyrase, indicating the presence of all enzymes necessary for glycolytic activity. The isozyme profiles of pyruvate kinase and malic enzyme, two enzymes involved in carbon metabolism during cleavage or in the subsequent activation of glycogen breakdown, do not change between the egg and gastrula stages. These data suggest that the activation of glycogen breakdown and glycolysis in the late blastula is probably not a result of new gene activity but may be the metabolic consequence of increased free ADP that is then able to support the pyruvate kinase reaction.
Mol Reprod Dev 1992 Aug
PMID:Glycogen breakdown in cleaving Xenopus embryos is limited by ADP. 149 83

Using the fluorescent indicators 2',7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein and Oxonol V to monitor intracellular pH (pHi) and cell membrane potential respectively, we have investigated the involvement of H(+)-dependent ATPase and H(+)-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24 mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pHi of 6.42. The subsequent recovery of pHi was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11 +/- 0.003 units, compared with 0.27 +/- 0.02 units under amiloride-free conditions. In the presence of the H(+)-dependent ATPase antagonist N,N'-dicyclohexylcarbodiimide (DCC), the pHi recovery observed in amiloride-containing, K(+)-free buffer was abolished. The recovery of pHi in Na(+)- and K(+)-containing buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pHi recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H(+)-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Endocrinol 1992 Feb
PMID:H(+)-dependent ATPase and K+ channel activities in the rat thyroid cell strain FRTL-5. 153 20

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Mar 05
PMID:X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments. 153 10

A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term "hash-marks" for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44/62 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hash-mark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.
J Mol Biol 1992 Mar 20
PMID:Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex. 153 38

We have cloned and sequenced a 5200 base restriction fragment and an overlapping 3100 base fragment of the large single copy region of the chloroplast genome of the diatom Odontella sinensis, which hybridized to several ATPase gene probes. These fragments contain six closely linked reading frames that were identified as atpI, atpH, atpG, atpF, atpD, and atpA, coding for subunits IV, III, II, I, delta, and alpha, respectively. Remarkably, the genes atpG and atpD, which are nucleus-encoded in chlorophyll a + b plants, are present in the Odontella chloroplast gene cluster. They map at the same positions as in cyanobacteria. The genes atpD and atpF overlap by four base-pairs as in certain photosynthetic and heterotrophic eubacteria. Upstream from the atpA gene cluster an open reading frame coding for 251 amino acid residues was found, which shows sequence similarity to ATP-binding subunits of periplasmic prokaryotic and eukaryotic transport systems. No similar reading frame is present in the land plant chloroplast genomes analysed so far. Sequences and arrangement of the genes are discussed with respect to the peculiar evolution of the chlorophyll a + c-containing chromophytic plastids.
J Mol Biol 1992 Mar 20
PMID:Chloroplast ATPase genes in the diatom Odontella sinensis reflect cyanobacterial characters in structure and arrangement. 153 39

Amastigotes of Leishmania donovani develop and multiply within the acidic phagolysosomes of mammalian macrophages. Isolated amastigotes are acidophilic; they catabolize substrates and synthesize macromolecules optimally at pH 5.5. Substrate transport in amastigotes has not been characterized. Here we show that amastigotes exhibit an uphill transport of proline (active transport) with an acid pH optimum (pH 5.5). It is dependent upon metabolic energy and is driven by proton motive force. Agents which selectively disturb the component forces of proton motive force, such as carbonyl cyanide chlorophenylhydrazone, nigericin and valinomycin, inhibit proline transport. Transport is sensitive to dicyclohexylcarbodiimide and insensitive to ouabain, demonstrating the involvement of a proton ATPase in the maintenance of proton motive force. It is suggested that the plasma membrane pH gradient probably makes the greatest contribution to proton motive force that drives substrate transport in the amastigote stage.
Mol Biochem Parasitol 1992 Mar
PMID:Proline transport in Leishmania donovani amastigotes: dependence on pH gradients and membrane potential. 153 14

The equilibrium distribution of tetraphenylphosphonium bromide was used to measure the membrane potential in Leishmania donovani amastigotes and promastigotes and to investigate mechanisms underlying the maintenance of membrane potential. At pH 7.0, membrane potential ranges between -90 and -113 mV. Increasing the external concentrations of hydrogen or potassium ions decreased membrane potential as did treatments with carbonylcyanide chlorophenylhydrazone or valinomycin. These observations are consistent with a membrane potential set by hydrogen and potassium ion diffusion gradients. Anaerobiosis lowered membrane potential, suggesting the involvement of ATPase(s) in maintaining membrane potential. Membrane potential was insensitive to treatment with ouabain, demonstrating the absence of a Na+/K(+)-ATPase. Treatment with dicyclohexylcarbodiimide caused a temporary hyperpolarization of the membrane suggesting the participation of a proton ATPase in the maintenance of membrane potential. Determination of the membrane potential makes it possible to quantitate the total proton motive force which is the force for active transport across the parasite membrane.
Mol Biochem Parasitol 1992 Mar
PMID:The plasma membrane electrical gradient (membrane potential) in Leishmania donovani promastigotes and amastigotes. 153 15

The 38 sequences of the ATPase c/III/9 gene determined in bacteria, fungi, mammals, and higher plants have been used to construct phylogenetic trees by distance matrix and parsimony methods (checked by bootstrapping); alignments have been performed on the deduced amino-acid sequences and then transferred back to the nucleotide sequences. Three lineages stand out: (1) eubacteria (except cyanobacteria and alpha purple bacteria), (2) chloroplasts, together with cyanobacteria, and (3) mitochondria together with nuclei and alpha purple bacteria. The clear monophyly of the mitochondrial/nuclear lineage, taken all together, strongly suggests that the nuclear copies of the gene now residing in the eukaryotic nucleus originate from a mitochondrial transfer. Within this lineage, metaphytes emerge late and as a cohesive group, after fungi (as a dispersed group) and metazoa, yielding an order that markedly differs from that obtained through typical RNA nuclear molecules. The possible biphyletic origin of mitochondria based on mitochondrial rRNA sequences is not evidenced by these sequences. Internal branches within both the chloroplastic and the mitochondrial lineages are consistent with botanical evolutionary schemes based on morphological characters. In spite of its relatively small size, the ATPase c/III/9 gene therefore displays remarkable properties as a phylogenetic index and adds a new tool for molecular evolutionary reconstructions, especially within the metaphytes.
J Mol Evol 1992 Apr
PMID:ATP synthase subunit c/III/9 gene sequences as a tool for interkingdom and metaphytes molecular phylogenies. 153 53

The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.
Mol Microbiol 1992 May
PMID:Biochemical activities of the parA partition protein of the P1 plasmid. 153 33

The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.
Mol Gen Genet 1992 Apr
PMID:A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments. 153 40


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