The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides average concentrations ±SD (μM) are: ATP, 3,152±1,698; GTP, 468±224; UTP, 567±460 and CTP, 278±242. For deoxynucleosidestriphosphate (dNTP), the concentrations in dividing cells are: dATP, 24±22; dGTP, 5.2±4.5; dCTP, 29±19 and dTTP 37±30. By comparison, dUTP is usually about 0.2 μM. For, the 4 dNTPs, tumor cells have concentrations of 6–11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2–5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4–6 μM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.

Physiological concentrations of purines and pyrimidines.

The Arabidopsis genome contains ∼200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR–encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

/pdf/genome-wide-analysis-of-nbs-lrr-encoding-genes-in-5eegxpfsdp.pdf

Genome-Wide Analysis of NBS-LRR–Encoding Genes in Arabidopsis

https://works.bepress.com/steven-whitham/22/download/

The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor

▪ Abstract In “gene-for-gene” interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.

/pdf/plant-disease-resistance-genes-3jj1d7brmf.pdf

Plant Disease Resistance Genes

Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2, 3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM, but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM.

Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases.

Analysis of a set of well characterized enzymes shows that the size of a protein subunit is directly related to the number of unique ligand binding functions described for the particular enzyme. The average size increment is about 5 000 Da per ligand binding function. This value corresponds very well to: (a) the amount of polypeptide chain required to form a stable folded structure, and (b) the size of polypeptide coded by the average exon. This reinforces the hypothesis that exon-coded modules are basic architectural units for proteins. Key predictive elements of this hypothesis are: 1) generally each module has a unique function, such as the ability to bind a specific ligand; 2) the size of an enzyme subunit should be determined by the number of modules required to accomplish the enzyme's biological role.

Are proteins made of modules

Uridine kinase: altered enzyme with decreased affinities for uridine and CTP.

Orotidine 5′-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis–Menten kinetics with Km = 350 ± 60 nM and Vmax = 2.70 ± 0.10 µmol/min/mg protein. Inhibition patterns for nucleoside 5′-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5′-monophosphate (Ki = 3.6 ± 0.7 nM) > xanthosine 5′-monophosphate (XMP, Ki = 4.4 ± 0.7 nM) > 6-azauridine 5′-monophosphate (AzaUMP, Ki = 12 ± 3 nM) > allopurinol-3-riboside 5′-monophosphate (Ki = 240 ± 20 nM). XMP is an ∼150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of ...

Thomas W. Traut

Papers

Are proteins made of modules

Uridine kinase: altered enzyme with decreased affinities for uridine and CTP.

Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum.

Effects of Substrate Binding Determinants in the Transition State for Orotidine 5′-Monophosphate Decarboxylase

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells