Lloyd R. Finch

The uptake of amino acids by isolated segments of rat intestine. II. A survey of affinity for uptake from rates of uptake and competition for uptake.

Average rates have been determined for the uptake by segments of rat small intestine of 17 amino acids at the concentrations of I mM and IO mM. At IO mM the order is such that the more lipophilic amino acids show the smaller rates, but at I mM the order is distinctly reversed, with the more lipophilic amino acids having the greater rates. On the assumption that Michaelis-Menten kinetics apply to the rate of uptake, Michaelis constants have been calculated from the average rates at I mM and IO mM. From these constants, assuming that the amino acids compete for uptake by a Michaelis-Menten mechanism, predictions have been made of the effect of one amino acid on the other and have been compared with observed values. In general, the predictions agree well with the observed values, suggesting that the assumptions may be correct; but exceptions to the agreement suggest that L-lysine, and probably L-ornithine and L-arginine, may not compete for a common mechanism with the other L-amino acids. If the agreement is taken to implicate a rate-limiting, reversible combination with a common site in the uptake of most L-amino acids, then such a site shows a higher affinity for the more lipophilic amino acids. Limited studies with D-amino acids suggest that they may also combine with the site for uptake of L-amino acids, but have a lower affinity for it.

Quantitative extraction and estimation of intracellular nucleoside triphosphates of Escherichia coli

Abstract Techniques for the estimation of intracellular nucleotides in Escherichia coli have been studied. The techniques have depended on the counting of [32P]-labeled nucleotides, separated by chromatography on PEI-cellulose thin layers after extraction from labeled cultures. Several methods of extraction have been examined in regard to the effect of duration of extraction and concentration or pH of extractant on the procedure. Using the optimum conditions indicated by this examination, comparisons have been made between methods. Our conclusion is that extraction with cold 0.4 M HClO4 is to be preferred over methods based on the use of hot water, cold trichloroacetic acid, sodium formate/formic acid mixtures, or acetic acid as the extractant. The method can be applied to unfiltered samples of culture if nucleotides are being estimated by incorporation of radioactive orthophosphate, provided that the preparation of radioactive orthophosphate is purified by ion-exchange chromatography before use.

The uptake of amino acids by isolated segments of rat intestine. I. A survey of factors affecting the measurement of uptake.

Abstract Techniques have been developed for the determination of the uptake of a large number of amino acids on a single preparation of segments from isolated small intestine of rats. The uptake can be followed by the fall in amino acid concentration of the external medium provided that a correction is made for water exchange between the tissue and the medium. As measured in this way the uptake may include both accumulation within, and metabolic transformation by the tissue. From recovery studies, metabolic transformation is likely to be of importance only in the uptake of l -ornithine, l -arganine, l -arpartic acid, l -glutamic acid and l -glutamine. The amino acids that are completely recoverable from the tissue after uptake show similar time-progresses of uptake with a cessation of net uptake suggestive of a steady state. The uptakes of all the amino acids, with the possible exceptions of l -arganine and l -lysine, are inhibited by 2,4-dinitrophenol. It is concluded that comparisons of rates of uptake may be made by determination of the uptake at 4 min.

New analytical procedure for the estimation of DNA with p-nitrophenylhydrazine

A new method for the colorimetric estimation of DNA has been developed by extensive modification of the Webb-Levy procedure using p-nitrophenylhydrazine as the colorimetric reagent. The method combines the chromogenic reaction with extraction of the DNA from the tissue, in order to minimize the destruction of DNA-deoxyribose that otherwise occurs during the extraction with hot acid. Other modifications include addition of sulfite to the reaction mixture, use of acetylated pNPH as the reagent, removal of excess reagent by reaction with acetylacetone, and extraction of the chromophore into butanol. These and other modifications have resulted in improved yield and stability of the chromophore and better reproducibility, in addition to the greater precision deriving from the combination of the processes of reaction and extraction of the DNA. The modified procedure gives a linear standard curve for 15 to 400 μg DNA in 4 ml reaction mixture and the upper limit of the range can be extended to at least 4 mg by dilution of a portion of the reaction mixture for chromophore development.

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells.

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.

Uridine‐5′‐monophosphate pyrophosphorylase activity from Escherichia coli

Uracil is readily incorporated into pyrimidine nucleotides and nucleic acids by many bacteria apparently via its reaction with 5-phosphoribosyl-l-pyrophosphate (PRPP) to give UMP [l-3]. While the enzyme catalysing this reaction has been detected [ 1,2] in extracts of Escherichia coli it has not been studied in detail. This information has not been available on kinetics or possible effecters of the enzyme. With dialysed crude extracts from E. coli we have found that the enzyme is strongly activated by GTP and inhibited by UMP and UTP. Preincubation with GTP is necessary for maximal activity and the specific activity of the extract decreases at extreme dilution. Uracil and PRPP show Michaelis-Menten kinetics as substrates with Km values of 4 X 1 O-6 M and 2 X 10m5 M respectively.

A simple centrifuge column for desalting protein solutions

Abstract Construction of a simple centrifuge column from readily available components is described which may be used for removing salt from protein solutions.

Enzymes of Intermediary Carbohydrate Metabolism in Ureaplasma urealyticum and Mycoplasma mycoides subsp. mycoides

Cell extracts of Ureaplasma urealyticum and Mycoplasma mycoides were examined for enzymes of intermediary carbohydrate metabolism using a sensitive radiochemical assay procedure. For M. mycoides, the enzyme activities detected were supporting evidence for the existence of a glycolytic pathway giving lactate anaerobically and acetate aerobically. U. urealyticum also had activities of many glycolytic enzymes. Enzymes of the pentose phosphate pathway occurred in both M. mycoides and U. urealyticum. Their presence allowed the proposal of a sequence for the synthesis from glycolytic pathway intermediates of ribose 5-phosphate, and hence phosphoribosyl diphosphate, for the synthesis of nucleotides. Pathways for the further metabolism of deoxyribose 1-phosphate and ribose 1-phosphate produced from nucleoside phosphorylase reactions operated in extracts from both organisms.

Characterization of a restriction endonuclease from Ureaplasma urealyticum 960 and differences in deoxyribonucleic acid modification of human ureaplasmas

Uur9601, a restriction endonuclease from Ureaplasma urealyticum 960T, cleaved at the sequence 5′-GC/NGC-3′ and is thus an isoschizomer of Fnu4HI. Fnu4HI cleaved deoxyribonucleic acid from human ureaplasma serovars I, III, and VI but not from II, IV, V, VII, VIII (strain 960), and IX. This grouping of serovars, indicative of their deoxyribonucleic acid modification, matches that previously reported by others using different criteria.

Inhibition of DNA synthesis and cell death

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.