Xanthosine-5'-phosphate amidotransferase from Escherichia coli.

The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate. This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase. The so-called XMP aminase from the E. coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase. These data demonstrate that this enzyme from E. coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.     

Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

Accumulation of Xanthosine by Escherichia coli in the Presence of Angustmycin C

Effect of angustmycin C, an adenosine analogue having an unusual sugar linked to adenine, on Escherichia coli was studied. It inhibited biosynthesis of RNA and DNA preferentially and xanthosine was excreted from the inhibited cells into the medium. During the course of its growth with addition of the antibiotic, the specific activity of inosine-5′-phosphate dehydrogenase of E. coli cells increased six times. These effects could be explained by the inhibitory effect of angustmycin C on xanthosine-5′-phosphate aminase which resulted in decreased level of IMP-dehydrogenase. Accumulation of xanthosine induced by the antibiotic reached the concentration of 940 μg/ml under an appropriate condition.     

Effect of vitamin B-12 deprivation on the rates of synthesis and degradation of rat liver fatty acid synthetase

To characterize the basis for the increased hepatic fatty acid synthetase activity in vitamin B-12 deprivation, the content and rates of synthesis and degradation for the enzyme were determined. Animals were in a dietary steady state on normal chow or a B-12-deprived diet; animals on the latter diet were further divided into a “supplemented” group given B-12 and those “B-12-deprived.” The B-12-deprived animals had tissue B-12 depletion. Both total and specific activity of fatty acid synthetase were increased in the B-12-deprived animals, and this was due to increased enzyme protein. Rates of synthesis and degradation were studied in each group after a pulse of 20 μCi of l-[U-¹⁴C]leucine. Radioactivity was determined in the immunoprecipitate of the purified enzyme. Relative rates of synthesis in the B-12-deprived group were increased 8.8-fold over the normal and 3.6-fold over the B-12-supplemented group. Degradation of hepatic fatty acid synthetase was 63 hr ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) in the normal and 65 hr in the B-12-supplemented group. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ in the B-12-deprived group was 35 hr. Degradation rates for the soluble protein pool were not affected by B-12 deprivation. The rate constant for synthesis of hepatic fatty acid synthetase in the B-12-deprived group was 14-fold that of the normal and 6-fold that of the B-12-supplemented animals. Thus, although vitamin B-12 deprivation results in increased rate of degradation of fatty acid synthetase, enzyme synthesis is markedly increased yielding a severalfold net increase in synthetase content and activity.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

5 (alpha-bromoacetyl)-2'=deoxyuridine 5'-phosphate: a mechanism based affinity label for thymidylate synthetase.

5(α-Bromoacetyl)-2′-deoxyuridine 5′-phosphate is an active site-directed irreversible inhibitor of thymidylate synthetase from $\text{Lactobacillus casei}$. The reversible inhibition (K_I4uM) is competitive with substrate and on incubation the reversible enzyme-inhibitor complex is converted to the irreversible complex with a first order rate constant (k₂) of 0.15 min^?1.     

Simple enzymatic procedure for preparation of 15N-labeled l-glutamic acid

The application of a number of immobilized Procion dyes to the purification of inosine 5′-monophosphate dehydrogenase (EC 1.2.1.14) from Escherichia coli has been investigated. The enzyme is adsorbed to a number of these immobilized dyes and can be eluted by salt gradients with very substantial increases in specific activity. For example, adsorption of the enzyme from a crude cell-free extract of E. coli to immobilized Procion yellow MX-8G in the presence of 15% ($\text{v}\text{v}$) ethylene glycol and subsequent elution with a salt gradient yields an enzyme preparation approximately 90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is quantitatively recovered with an overall increase in specific activity of 14-fold compared to the enzyme in the cell-free extract.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.     

Stimulation by cyclic nucleotides of prostaglandin E production in isolated Graafian follicles

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5′ AMP and 8-Br-5′ GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors.Administration of hCG $\text{in vivo}$ or incubation with LH $\text{in vitro}$ did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG.Collectively, the results suggest that stimulation of ovarian PGE production by cyclic mucleotides and LH is dependent on $\text{de novo}$ synthesis of one more components of the PG synthetase systme rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Regulation of pyridoxal 5'-phosphate metabolism in liver

The pyridoxal 5′-phosphate content of liver $\text{in vivo}$ and of hepatocytes $\text{in vitro}$ remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.     

Adaptive synthesis of rat liver fatty acid synthetase: evidence for in vitro formation of active enzyme from inactive protein precursors and 4'-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated $\text{in vitro}$ when CoA or $\text{E. coli}$ acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When (¹⁴C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.     

5-Nitro-2'-deoxyuridine 5'-monophosphate is a potent irreversible inhibitor of Lactobacillus caesi thymidylate synthetase.

5-Nitro-2′-deoxyuridine 5′-monophosphate was found to be an active sitedirected irreversible inhibitor of thymidylate synthetase from $\text{Lactobacillus caesi}$. It's K_I was determined as 2.9 × 10^?8$\text{M}$ from a double-reciprocal plot of velocity $\text{vs}$ substrate concentration.     

Separation of pigeon liver apo- and holo- fatty acid synthetases by affinity chromatography.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [¹⁴C]pantetheine-labeled fatty acid synthetase complex [Lornitzo $\text{et al.}$, J. Biol. Chem. $\text{249}$, 1654 (1974)]. When [¹⁴C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [¹⁴C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3'-5'-cyclic AMP

A determination of the level of the pyruvate dehydrogenase complex in a 3′–5′-c-AMP deficient mutant of $\text{E.}$$\text{coli}$ K12 has been carried out. The deficiency has no effect on specific activities for derivatives carrying either the inducible genes for two components of the complex or constitutive mutants. We conclude that synthesis of the complex is not sensitive to catabolite repression.     

The extraction of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation.

It has been found that the most widely used method for the extraction of guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) from $\text{E. coli}$ (1 M formic acid at 0°) results in its $\text{in vitro}$ degradation to ppGp and GDP. A comparison with several other extraction procedures indicated that this breakdown is due to the low pH of the reagents used during extraction. This degradation can largely be prevented by using a new extraction technique which involves freezing and thawing of the cells in the presence of lysozyme at a neutral pH followed by treatment with deoxycholate. With this method it is possible to recover from three to five times as much ppGpp from both unstarved and amino acid starved stringent strains of $\text{E. coli}$ as compared with the most widely used formic acid procedure. Consequently, it will be necessary to reevaluate the ppGpp values obtained from cells when formic acid or other low pH reagents were used during extraction.     

Synthesis and properties of a new fluorescent analog of ATP: Adenosine-5′-triphosphoro-γ-1-(5-sulfonic acid) napthylamidate

An analog of ATP has been synthesized which contains the fluorophore, 1-aminonapthalene-5-sulfonate attached via a γ-phosphoamidate bond. This analog is strongly fluorescent (quantum yield = 0.63) with an emission maximum at 460 nm; the excited state lifetime is 20 nsec. It is a substrate for DNA-dependent RNA polymerase of $\text{E. coli}$ and wheat germ RNA polymerase II. It is also a substrate for $\text{E. coli}$ valyl t-RNA synthetase, venom phosphodiesterase, and potato apyrase. Cleavage of the α-β phosphoryl bond as a result of RNA synthesis or by venom phosphodiesterase produces a 15 nm red shift in the fluorescence emission spectrum. This property should make this nucleotide useful for studies of the mechanisms of enzymatic reactions involving cleavage of the α-β phosphoryl bond.