Phosphate transport in Micrococcus lysodeikticus.

Phosphate accumulates in Micrococcus lysodeikticus cells against a concentration gradient, by an energy-dependent process. The phosphate transport is derepressed during phosphate deprivation. The depression process is inhibited by chloramphenicol. The apparent Km of phosphate transport is 4.3 micronM. The activation energy of the transport is 21 kcal per mol in the temperature range of 0-29degrees C, and 4.9 kcal per mol between 29 and 40degrees C. The rate of the transport increases in presence of K+ and Mg2+. Arsenate is a competitive inhibitor of phosphate transport, having an apparent Ki of 6.0 micronM. Sulfhydryl reagents, respiratory inhibitors and uncouplers of oxidative phosphorylation inhibit phosphate transport.

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts.

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (delta psi), determined according to the distribution of the cation tetraphenylphosphonium (TPP+) between the cells and the medium. Reduction of 26, 18 and 13 mV in delta psi was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH2]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of delta psi, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the beta, gamma-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that delta psi is involved in the control of membrane permeability.

Effects of salts and ionophores on proline transport in a moderately halopholic halotolerant bacterium.

The effect of salt on proline uptake in a moderately halophilic halotolerant bacterium was studied. Cells were grown either on low salt or high salt media. A correlation was found between the salt concentrations in the growth media and the optimal concentration for uptake. The uptake rate was stimulated 2--3-fold by NaCl, as compared to KCl. The Km, V and activation energies values for proline uptake, as well as the external pH effect, were similar in low-salt-grown cells and high-salt-grown cells. This suggests that the halotolerance of the transport system is not due to alterations of the system during growth at various conditions, but rather to its intrinsic ability to function under extreme environmental conditions. The uptake was inhibited by cyanide and carbonyl cyanide m-chlorophenylhydrazone, but not by arsenate, indicating that the electrochemical proton gradient (delta mu- H+), generated by respiration, is the main driving force for proline transport. In low-salt-grown cells, at pH 6.0, partial inhibition was exerted by nigericin or valinomycin, whereas at pH 8.0 the uptake was inhibited by valinomycin only. Similar, although less pronounced effects were found in high-salt-grown cells. The data suggest that at pH 6.0 proline transport is driven by delta mu- H+ (composed of electrical potential (delta psi) and pH gradient), whereas at pH 8.0 delta psi is the main driving force. Procedures of pretreatment with EDTA were developed to enable the penetration of the ionophores into the cells.

Membrane-bound ATPase in chloroplasts of Euglena gracilis.

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca2+- and Mg2+-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. Km values of Ca2+ and Mg2+ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca2+- or Mg2+-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca2+ or Mg2+ or from two different enzymes are discussed.

Evaluation of PSI-BLAST alignment accuracy in comparison to structural alignments.

The PSI-BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI-BLAST alignments on a stringent database of 123 structurally similar, sequence-dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI-BLAST. Whenever a pair member detects its pair-mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the percentage of these positions out of the structural alignment. Fifty-two sequences detected their pair-mates (for 16 pairs the success was bi-directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5+/-2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9+/-2.5% within the five iterations tested in the current study.

Combined effects of ATP and its analogs on the membrane permeability in transformed mouse fibroblasts.

Extracellular ATP (0.6 mM) induces a marked decrease in the membrane potential, followed by an increase in cell membrane permeability in transformed mouse fibroblasts. The effects of the ATP analogs, p[CH2]ppA and p[NH]ppA (0.6 mM), on the membrane potential and permeability are much less pronounced. ATP at 0.05 mM has no effect by itself, but markedly increases the analog-induced membrane potential dissipation and permeability. The data suggest that ATP-induced membrane permeation is composed of two processes: One is common to ATP and its analogs and appears to be a receptor-mediated process. The second is unique for ATP, effective even at low concentration (0.05 mM), and might be mediated by cell surface enzymes, for which ATP, but not its analogs, serves as a substrate.

Computational protein function prediction: are we making progress?

The computational prediction of gene and protein function is rapidly gaining ground as a central undertaking in computational biology. Making sense of the flood of genomic data requires fast and reliable annotation. Many ingenious algorithms have been devised to infer a protein's function from its amino acid sequence, 3D structure and chromosomal location of the encoding genes. However, there are significant challenges in assessing how well these programs perform. In this article we explore those challenges and review our own attempt at assessing the performance of those programs. We conclude that the task is far from complete and that a critical assessment of the performance of function prediction programs is necessary to make true progress in computational function prediction.

Localization of phosphoglucose isomerase in Escherichia coli and its relation to the induction of the hexose phosphate transport system.

The localization of phosphoglucose isomerase (PGI) was studied in relation to the induction of hexose phosphate uptake in Escherichia coli. The uptake system is induced only by extracellular glucose-6-phosphate (G6P); there is no induction by intracellular G6P. Fructose-6-phosphate (F6P) is an indirect inducer, and isomerization of F6P to G6P must occur before induction. PGI has been considered to be an internal enzyme; therefore, uptake of F6P by noninduced cells and leakage of the G6P formed would be required for induction. In this study, it was concluded that part of the PGI activity is located in the cell surface because: (i) uninduced, intact cells are able to convert F6P to G6P, whereas the activity of G6P dehydrogenase is not detectable; (ii) when cells are subjected to osmotic shock, about 10% of the PGI activity is found in the shock fluid; and (iii) sorbitol-6-phosphate (S6P) inhibits both PGI activity of whole cells and the induction of hexose phosphate transport system by F6P. S6P was not taken by intact cells. The data indicate that the isomerization of F6P to G6P can take place on the cell surface, and this explains the indirect induction of hexose phosphate transport by F6P.

Recombinant in vitro tools to predict drug metabolism and safety.

Drug metabolism determines several pharmacological and toxicological properties of pharmaceuticals and is catalysed by drug metabolizing enzymes. Prediction of drug metabolism in humans based on animal experiments is complicated by species differences in the catalytic properties of these enzymes. This review describes and evaluates the use of recombinant models that contain human drug metabolizing enzymes to facilitate the prediction of pharmacokinetic properties of candidate drugs in humans.

Structures containing polyphosphate in Micrococcus lysodeikticus.

Granular structures containing inorganic polyphosphate were found in Micrococcus lysodeikticus. These structures were isolated by fractionation of the bacterial extract obtained by lysing the organisms with lysozyme. The composition of the fraction which was enriched with these structures was found to be: protein, 24%; lipids, 30%; and polyphosphate, 27%. This fraction also contained small amounts of ribonucleic acids, carbohydrate, and polyvalent cations. The effect of different reagents and enzymes on the integrity of the granules was examined. It was noticed that they accumulate in the bacteria during the logarithmic phase of growth but disappear gradually during the stationary phase.

Respiratory control in Micrococcus lysodeikticus.

The respiration rate of Pi-deprived cells of Micrococcus lysodeikticus is markedly increased by Pi, and returns to the original level following Pi consumption. The stimulation of the respiration was found to be specific for Pi and arsenate. Although succinate and valinomycin enhanced the respiration of both Pi-grown and Pi-deprived cells, only the latter could be further stimulated by Pi. The effect of Pi on the respiration rate was found to be concentration dependent. The control of respiration by Pi is due to its rapid uptake and its subsequent polymerization to polyphosphate via ATP. Both of these processes are coupled to proton influx into the cell, and thus stimulate the proton efflux and the respiration rate.

Heat-induced alterations in cell membrane permeability and cell inactivation of transformed mouse fibroblasts.

Hyperthermia, has recently been extended in many permutations as a modality of anticancer treatment, but the mechanisms underlying heat-induced cell inactivation are poorly understood. In this study, the role of the cell permeability barrier in the process of heat cytotoxicity are examined. Changes in cell membrane permeability were determined by following the efflux of normally impermeant metabolites, e.g. nucleotides, in cultures of Swiss mouse 3T3 cells, and their transformed derivatives, 3T6 cells. The increase in cell membrane permeability as a function of temperature and exposure duration was found to be characterized by a sigmoid curve, with a threshold value, above which the permeability markedly increased. A correlation was found between cell membrane permeabilization and cell inactivation. Both heat-induced permeabilization and heat cytotoxicity were more pronounced in the transformed cells, as compared to their untransformed counterparts. The temperature-dependent permeabilization was more effective in the presence of the ionophore amphotericin B. The data suggest that heat-induced lesion in the cell membrane has a major role in hyperthermia cytotoxicity.

ATP-resistant variants of transformed mouse fibroblasts.

Addition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non-transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 1.0 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP-resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non-transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP-induced alterations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.

Growth inhibition of breast cancer cells induced by exogenous ATP.

Addition of ATP (> 0.1 mM) to cultures of human breast cancer T47D cells resulted in an inhibition of cell proliferation. The inhibition was found to be specific for ATP, and dependent on its concentration. Growth inhibition continued for at least three days, although ATP and its hydrolysis products were metabolized within one day. Conditioned medium from ATP-treated cultures (CM+) was found to inhibit the growth of cells that were not exposed to ATP. This is an indication that extracellular factors, besides ATP, are involved in the inhibition process. The inhibition was maintained after dialysis of the CM+, using an 8 kDa cut-off membrane. Conditioned medium from untreated cultures (CM-), however, only slightly affected cell growth. The data suggest that the CM(+)-induced cell growth inhibition is mediated by an ATP-activated growth inhibiting factor. Flow microfluorometry and thymidine incorporation experiments have shown that the growth arrest is mainly due to the elongation of the S-phase of the cell cycle.