Mass spectrometric evidence for the modification of small molecules in a cobalt-60-irradiated rodent diet.

The purpose of this study was to investigate the effect of radiation on the content of animal diet constituents using global metabolomics. Aqueous methanolic extracts of control and cobalt-60-irradiated Teklad 7001 diets were comprehensively analyzed using nano-liquid chromatography-MS/MS. Among the over 2000 ions revealed by XCMS followed by data preprocessing, 94 positive and 143 negative metabolite ions had greater than 1.5-fold changes and p-values <0.01. Use of MetaboAnalyst statistical software demonstrated complete separation of the irradiated and non-radiated diets in unsupervised principal components analysis and supervised partial least squares discriminant analysis. Irradiation led to an increase in the content of phytochemicals such as glucosinolates and oxidized lipids in the diet. Twenty-eight metabolites that were significantly changed in the irradiated samples were putatively identified at the level of molecular formulae by MS/MS. MS/MSALL analysis of chloroform-methanol extracts of the irradiated diet showed increased levels of a number of unique linoleic acid-derived branched fatty acid esters of hydroxy fatty acids. These data imply that gamma irradiation of animal diets causes chemical changes to dietary components, which in turn may influence the risk of mammary cancer. Copyright © 2017 John Wiley & Sons, Ltd.

Isoflavone metabolism in domestic cats (Felis catus): comparison of plasma metabolites detected after ingestion of two different dietary forms of genistein and daidzein.

Some felid diets contain isoflavones but the metabolic capacity of cats toward isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet (n = 6) or as part of a dietary matrix (n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and disulfate as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and disulfate, dihydrodaidzein, and O-desmethylangolensin. No ß-glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared with cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein and daidzein, in domestic cat plasma after dietary ingestion has been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of typical dietary intake.

Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149.

The steady-state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH. The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d(7) by these enzymes have also been measured. The results show that the activity of wild-type GalE is pH-independent in the pH range of 5.5-9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d(7). It is concluded that the rate-limiting step for epimerization by wild-type GalE is not hydride transfer and must be either a diffusional process or a conformational change. Epimerization of UDP-galactose-C-d(7) by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on k(cat) of 2.2 +/- 0.4 at pH 6.2 and 1.1 +/- 0.5 at pH 8.3. Moreover, the plot of log k(cat)/K(m) breaks downward on the acid side with a fitted value of 7.1 for the pK(a). It is concluded that the break in the pH-rate profile arises from a change in the rate-limiting step from hydride transfer at low pH to a conformational change at high pH. Epimerization of UDP-galactose-C-d(7) by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on k(cat) of 2.0 +/- 0.2 between pH 6 and 9. Both plots of log k(cat) and log k(cat)/K(m) display pH dependence. The plot of log k(cat) versus pH breaks downward with a pK(a) of 6.35 +/- 0.10. The plot of log k(cat)/K(m) versus pH is bell-shaped, with fitted pK(a) values of 6.76 +/- 0.09 and 9.32 +/- 0.21. It is concluded that hydride transfer is rate-limiting, and the pK(a) of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pK(a) when measured spectrophotometrically in this variant. Acid-base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149. The kinetic pK(a) of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent kinetic isotope effect. Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at a k(cat) that is lower by a factor of 10(7) than that of wild-type GalE. This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild-type GalE and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme.

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159.

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pKa of 8.4 +/- 0.1. The simplest interpretation is that the apparent pKa is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0. 0213 at 25 degrees C, corresponding to DeltaG'o = +2.3 kcal mol-1. From this value and the standard free energy change of -10.9 kcal mol-1 for the hydrolysis of ATP to AMP and PPi, we calculate that DeltaG'o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol-1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pKa of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the alpha,beta-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.

Lack of biocytin transfer at gap junctions in the chicken vestibular nuclei.

In vivo experiments were designed to test for functional gap junctions at 'mixed' synapses that were morphologically characterized between the large-diameter, primary vestibular fibers and second-order vestibular neurons in the chicken, Gallus gallus. In previous intracellular recordings and dye injections into these neurons from brain slice preparations of chick embryos (E15/16) and also newborn hatchlings (HI-2), no evidence was obtained for functional gap junctions. Therefore, biocytin, a low molecular weight tracer that permeates gap junction channels, was extracellularly applied to either the ampullary nerves or to the vestibular ganglion of 3-6 day old hatchlings and adult chickens (9 months). This procedure resulted in the uptake of the dye and heavy staining of both the thick and thin fibers composing the vestibular nerve and in loading of vestibular efferent neurons. However, no dye transfer was observed between the large-diameter, primary vestibular fibers and second-order vestibular neurons. This observation, which was performed using a relatively non-invasive approach on intact animals, suggests that the gap junctions at these mixed synapses are probably not functional under the conditions of these experiments.

Standard free energies for uridylyl group transfer by hexose-1-P uridylyltransferase and UDP-hexose synthase and for the hydrolysis of uridine 5'-phosphoimidazolate.

The reversible reaction of UDP-glucose with imidazole (Im) to produce uridine 5'-phoshoimidazolate (UMPIm) and glucose-1-P is catalyzed by UDP-hexose synthase, which is the mutant H166G of hexose-1-P uridylyltransferase (EC 2.7.7.12) [Kim, J., Ruzicka, F.J., & Frey, P.A. (1990) Biochemistry 29, 10590-10593]. The availability of UDP-hexose synthase allows the equilibrium constant for the reaction UDP-glucose + Im = UMPIm + glucose-1-P to be measured, and it is found to be 2.2 x 10(-2) at pH 8.5 and 27 degrees C. At pH 7.0 and 27 degrees C the equilibrium constant is 6.4 x 10(-4). The equilibrium constant for the formation of the covalent uridylyl-enzyme intermediate of hexose-1-P uridylyltransferase (E-His(166) + UDP-glucose = E-His(166)-UMP + glucose-1-P) is found to be 1.8 x 10(-4) at pH 7.0 and 25 degrees C, which is slightly less favorable than the formation of UMPIm from UDP-glucose and Im. These equilibrium constants, when considered in the light of other data in the literature, allow the standard free energy changes for the hydrolysis of UMPIm and the analogous covalent uridylyl-enzyme intermediate to be calculated. The results show that delta G' degrees (delta G degrees (ph)(7.0)) for the hydrolyses of UMPIm and E-His(166)-UMP are -14.7 and -15.4 kcal mol(-1), respectively at pH 7.0. At pH 8.5, the corresponding values of delta G degrees (ph) (8.5) are -12.6 and -9.9 kcal mol(-1), respectively. It is concluded that noncovalent binding interactions between the active site and the UMP group of E-His(166)-UMP provide little or no stabilization in the formation of this species as an intermediate in the reaction of hexose-1-P uridylyltransferase.

Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase.

A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid. The fusion enzyme was expressed in E. coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose. On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities. The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes. The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.

A simplified procedure for synthesizing nucleoside 1-thiotriphosphates: dATP alpha S, dGTP alpha S, UTP alpha S, and dTTP alpha S.

A procedure for synthesizing a nucleoside 1-thiotriphosphate in a single reaction vessel beginning with the nucleoside, PSCl3, and PPi is described. Reaction of the dried nucleoside with PSCl3 in anhydrous triethylphosphate is followed by addition of the tetrabutylammonium salt of PPi. Addition of excess triethylamine precipitates the nucleotides. The crude product is dissolved in water, and the nucleoside 1-thiotriphosphate is purified in 24% to 50% yield by chromatography. The method is applied to the synthesis of dATP alpha S, dGTP alpha S, UTP alpha S and dTTP alpha S.

Uridine diphosphate galactose 4-epimerase. pH dependence of the reduction of NAD+ by a substrate analog.

Neutron activation analysis of UDP-galactose 4-epimerase from Escherichia coli for 53 metals shows that the enzyme does not contain any of these metals at significant levels. The substrate analog P1-5'-uridine-P2-glucose-6-yl pyrophosphate (UGP), a structural isomer of UDP-glucose with the sugar linked to UDP through the C-6 hydroxyl group, is an inactivator that irreversibly reduces epimerase.NAD+ to epimerase.NADH. The pH dependence of kobs reveals the essential involvement of an acidic group, kinetically measured pKa = 5.48 +/- 0.08, in unprotonated form and two weakly acidic or basic groups, apparent pKa values of 10.03 +/- 0.43, in protonated forms. Measurements of kobs as a function of [UGP] show that it is given by kobs = k[UGP]/(K + [UGP]) at a given pH, where K = 0.19 +/- 0.04 mM throughout the pH range 4.8-10.4. The pH-dependent first order rate constants range from 0.28 to 1.94 s-1, with the maximum value at pH 7.6 and decreasing at acidic and basic pH values. Reaction of [glucose-1-2H]UGP proceeds with kinetic isotope effects of 5.0, 2.1, 2.0, 1.9, and 3.5 at pH values 5.0, 6.2, 7.6, 9.0, and 10.0, respectively. Therefore, hydride transfer becomes rate-limiting at pH extremes but is not limiting at neutral pH, although deuteride transfer is significantly limiting at all pH values. The isotope effects facilitated correction of the kinetic pK values to the thermodynamic values 6.1-6.2 on the acid side and 9.0-9.6 on the alkaline side. We postulate that the group with pK1 = 5.5 (6.1-6.2 corrected) functions as an enzymic general base that abstracts the glucosyl C-1 hydroxyl proton in concert with transfer of the C-1 hydrogen and two electrons to NAD+. The pH dependence on the alkaline side may be related to the uridine nucleotide-dependent conformational transition that is an essential step in the reduction of epimerase.NAD+ to epimerase.NADH by sugars.

Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism.

A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)