Cross-linking of two beta subunits in the closed conformation in F1-ATPase.
In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP. (+info)
Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells.
Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water. (+info)
Regulation of a volume-sensitive anion channel in rat pancreatic beta-cells by intracellular adenine nucleotides.
1. The patch-clamp technique in the whole-cell configuration was used to measure the effects of intracellular adenine nucleotides on activity of the volume-sensitive anion channel in single, isolated rat pancreatic beta-cells. 2. In the absence of intracellular nucleotides, swelling of cells with a hypertonic pipette solution failed to activate the conductance. Addition of ATP over the range 2-10 mM maintaining the same degree of hypertonicity caused a progressive activation of the conductance. An increase in ATP produced a similar activation of the conductance in non-swollen cells, albeit with reduced current amplitudes. 3. Activation of the conductance was also observed in the presence of ATPgammaS, adenylyl imidophosphate (AMP-PNP), ADP, diadenosine tetraphosphate and GTPgammaS. Neither ADP nor GDPbetaS inhibited activation of the conductance by ATP. 4. It is concluded that activity of the beta-cell volume-sensitive anion channel can be modulated by changes in intracellular concentrations of ATP within the physiological concentration range by a mechanism that does not require nucleotide hydrolysis. Activity of the channel does not appear to be modulated by a G protein-coupled mechanism. (+info)
Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.
In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis. (+info)
Hypotension induced by exercise is associated with enhanced release of adenyl purines from aged rat artery.
To determine whether the antihypertensive effects of exercise are associated with release of ATP and its metabolites from arteries, we assayed blood pressure and the release of adenine nucleotides and nucleosides from the caudal arteries of exercised and sedentary aged hypercholesterolemic rats. Exercise on a treadmill for 12 wk significantly decreased the rise in systolic and diastolic blood pressure by 7.5 and 15.9%, respectively, with advanced age. The concentrations of oleic, linoleic, and linolenic acids in the caudal artery decreased significantly with exercise, demonstrating an association between exercise and the unsaturation index of caudal arterial fatty acids. The amounts of total adenyl purines released by the arterial segments from exercised rats, both spontaneously and in response to norepinephrine, were significantly greater by 80.0 and 60.7%, respectively, than those released by tissues from sedentary rats. These results suggest that exercise alters the membrane fatty acid composition in aged rats as well as the release of ATP from vascular endothelial cells and that these factors are associated with the regression of the rise in blood pressure normally observed with advanced age. (+info)
Effect of zinc on adenine nucleotide pools in relation to aflatoxin biosynthesis in Aspergillus parasiticus.
The adenylic acid systems of Aspergillus parasiticus were studied in zinc-replete and zinc-deficient media. The adenosine 5'-triphosphate levels of the fungus were high during exponential phase and low during stationary phase in zinc-replete cultures. On the other hand, the levels of adenosine 5'-diphosphate and adenosine 5'-monophosphate were low during exponential phase of growth and high during stationary phase. The adenosine 5'-triphosphate levels during exponential phase may indicate higher primary metabolic activity of the fungus. On the other hand, high adenosine 5'-monophosphate levels during stationary phase may inhibit lipid formation and may enhance aflatoxin levels. The inorganic phosphorus content was low in a zinc-replete medium throughout the growth period, thereby favoring aflatoxin biosynthesis. The energy charge during the exponential phase was high but low during the stationary phase. In general the energy charge values were lower because of high adenosine 5'-monophosphate content. (+info)
Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.
A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism. (+info)
Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR.
The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism. (+info)