inosine-triphosphate and thymidine-5--triphosphate

The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

Topics: Adenosine Triphosphate; Bacterial Proteins; Burkholderia; Catalysis; Cations, Divalent; Cloning, Molecular; Enzyme Stability; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inosine Triphosphate; Kinetics; Molecular Weight; Phosphofructokinases; Phosphotransferases; Recombinant Proteins; Substrate Specificity; Thymine Nucleotides

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Dideoxyadenosine; DNA Primers; DNA-Directed DNA Polymerase; Inosine Triphosphate; Polymerase Chain Reaction; Potassium Chloride; Quality Control; Sensitivity and Specificity; Sequence Analysis, DNA; Templates, Genetic; Thymine Nucleotides

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

Topics: Adenosine Triphosphate; Ambystoma; Animals; Barium; Biological Transport; Cations; Cells, Cultured; Cytidine Triphosphate; Diazoxide; Diuretics; Electric Conductivity; Glyburide; Guanosine Triphosphate; Hypoglycemic Agents; Inosine Triphosphate; Ion Channel Gating; Kidney Tubules, Proximal; Kinetics; Microvilli; Patch-Clamp Techniques; Potassium Channels; Sodium Chloride Symporter Inhibitors; Sodium-Potassium-Exchanging ATPase; Thallium; Thymine Nucleotides; Uridine Triphosphate