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W J Gray

Publications and source records attributed to W J Gray.

32 records · Page 2Linked to original sources
PubMed1982

Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms.

The molecular weight of the purple, iron-containing glycoprotein uteroferrin has been determined to be 35140 +/- 1610 by sedimentation equilibrium centrifugation. This is consistent with measurements carried out by other procedures. The dry weight of a solution of uteroferrin with an absorption of 1.00 cm -1 at 280 nm was 0.87 mg/ml. Highly purified uteroferrin has a ratio of absorbance at 280 nm to 545 nm of about 13.2 and a revised extinction coefficient at 545 nm of 3.1 . 10(3) M -1 is presented. The iron content of uteroferrin has been determined both colorimetrically and by atomic absorption spectroscopy. One iron atom is present per polypeptide chain. Reduction with dithionite leads to loss of iron, allowing the apoprotein to be prepared. Enzymatic activity can be restored to the apoprotein with Fe(III) or with Cu(II). The copper enzyme has no visible color and has a higher pH optimum than the Fe-uteroferrin. Whereas the phosphatase activity of the latter is increased several-fold by beta-mercaptoethanol, reduction inactivates Cu-uteroferrin. Both forms of uteroferrin have similar Km values for p--nitrophenyl phosphate and are inhibited by molybdate but not by tartrate. Excess Cu(II) and Fe(III) strongly inhibit uteroferrin phosphatase activity and these results may explain the failure of other to restore activity to the apoprotein using Cu(II) and Fe(III) salts.

Acid Phosphatase↗
PubMed1972

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli.

The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

Amino Acids↗
PubMed1972

The control of ribonucleic acid synthesis in bacteria. Polymerization rates for ribonucleic acids in amino acid-starved relaxed and stringent auxotrophs of Escherichia coli.

Polymerization rates of newly formed chains of various RNA fractions were measured in Escherichia coli CP78 (RC(str)) and CP79 (RC(rel)) multiple amino acid auxotrophs, deprived of four amino acids essential for growth. Immediately after the onset of severe amino acid deprivation, in RC(str) strains the rate of labelling of RNA by exogenous nucleotide bases was greatly diminished. At first, the initiation of new RNA chains declined faster than the rate of polymerization in RC(str) organisms, but as starvation proceeded the rate of polymerization was eventually lowered to about 10% of that found during normal growth. In strain CP79 (RC(rel)), on the other hand, chain-polymerization rates were unaffected by amino acid withdrawal. Artificial depletion of the intracellular purine nucleotide pools in RC(str) or RC(rel) strains by trimethoprim, before the onset of amino acid deprivation, showed that in the RC(str), but not the RC(rel) strain, amino acid withdrawal gave rise to an inability of the cells to utilize exogenously supplied purine or pyrimidine bases for RNA synthesis. During a prolonged starvation, the observed 100-fold decrease in the total rate of incorporation of exogenous nucleotide bases into the RNA of RC(str) organisms was ascribed to a combination of a tenfold decrease in the overall rate of RNA chain polymerization, at least a fivefold decrease in the ability of the cells to utilize exogenous bases and a preferential inhibition of initiation of stable RNA chains. None of these changes occurred in the corresponding RC(rel) strain.

Adenine↗
PubMed1971

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in chloramphenicol-inhibited cultures of Escherichia coli.

The rate of polymerization of ribosomal ribonucleic acid chains was estimated for steadily growing cultures of Escherichia coli M.R.E.600, from the kinetics of incorporation of exogenous [5-(3)H]uracil into completed 23S rRNA molecules. The analytical method of Avery & Midgley (1971) was used. Measurements were made at 37 degrees C, in the presence or the absence of chloramphenicol, in each of three media; enriched broth, glucose-salts or sodium lactate-salts. The rate of chain elongation of 23S rRNA was virtually constant in all media at 37 degrees C, as 24+/-4 nucleotides added/s. Accelerations in the rate of biosynthesis of rRNA by chloramphenicol in growth-limiting media are due primarily to an increase in the rate of initiation of new RNA chains, up to the rates existing in cultures growing rapidly in broth. Thus, in poorer media, only a small fraction of the available DNA-dependent RNA polymerase molecules are active at any given instant, since the chain-initiation rate is limiting in these conditions. In cultures growing rapidly in enriched broth, antibiotic inhibition caused a rise of some 12% in the rate of incorporation of exogenous uracil into total RNA. This small acceleration was due entirely to the partial stabilization of the mRNA fraction, which accumulated as 14% of the RNA formed after the addition of chloramphenicol. In cultures growing more slowly in glucose-salts or lactate-salts media, chloramphenicol caused an immediate acceleration of two- to three-fold in the overall rate of RNA synthesis. Studies by DNA-RNA hybridization showed that the synthesis of mRNA was accelerated in harmony with the other affected species. However, just over half the mRNA formed after the addition of chloramphenicol quickly decayed to acid-soluble products, whereas the remainder was more stable and accumulated in the cells. The mRNA fraction constituted about 6% of the total cellular RNA after 3h inhibition. A model was suggested to explain the partial stabilization and accumulation of the mRNA fraction and the acceleration in the rate of synthesis of mRNA when chloramphenicol was added to cultures in growth-limiting media.

Bacterial Proteins↗
PubMed1971

The control of ribonucleic acid synthesis in bacteria. The synthesis and stbility of ribonucleic acid in rifampicin-inhibited cultures of Escherichia coli.

A study was made of the kinetics of labelling of the stable ribonucleic acids (rRNA+tRNA) and the unstable mRNA fraction in cultures of Escherichia coli M.R.E.600, inhibited by the addition of 0.1g of rifampicin/l. Labelling was carried out by adding either [2-(14)C]- or [5-(3)H]-uracil as an exogenous precursor of the cellular nucleic acids. From studies using DNA RNA hybridization, the kinetics of the synthesis and degradation of mRNA was followed in the inhibited cultures. Although a considerable proportion of the mRNA labelled in the presence of rifampicin decayed to non-hybridizable products, about 25% was stabilized beyond the point where protein synthesis had finally ceased. It therefore seems unwise to extrapolate the results of studies on mRNA stability in rifampicin-inhibited cultures to the situation existing in the rate of steady growth, where there appears to be little, if any, stable messenger. The kinetics of labelling of RNA in inhibited cultures indicated that the clapsed time from the addition of rifampicin to the point at which radioactivity no longer enters the total cellular ribonucleic acids is a measure of the time required to polymerize a molecule of rRNA. At 37 degrees C, in culture grown in broth, glucose-salts or lactate salts media, exogenous [2-(14)C]uracil entered rifampicin-inhibited cells and was incorporated into RNA for 2 3min after the antibiotic was added. Taking this time as that required to polymerize a complete chain of 23S rRNA, the polymerization rate of this fraction in the three media was 25, 22 and 19 nucleotides added/s to the growing chains. Similar experiments in cultures previously inhibited by 0.2g of chloramphenicol/l showed virtually identical behaviour. This confirmed the work of Midgley & Gray (1971), who, by a different approach, showed that the polymerization rate of rRNA in steadily growing and chloramphenicol-inhibited cultures of E. coli at 37 degrees C was essentially constant at about 22 nucleotides added/s. It was thus confirmed that the rate of polymerization of at least the rRNA fraction in E. coli is virtually unaffected by the nature of the growth medium and therefore by bacterial growth rate.

Bacterial Proteins↗
PubMed1970

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600.

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.

Cell Division↗