Robert J. Roon

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

Utilization of the resolved L-isomer of 2-amino-6-phosphonohexanoic acid (L-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons.

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the α-amino-ω-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereasl-AP4 andd-AP5 cross-react with other EAA receptors,dl-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity ofdl-AP6, in conjuction with the apparent preference of this site forl-isomers, suggested that the hitherto unavailablel-isomer of AP6 would be a potent and specific agonist. We report the resolution of thed- andl-enantiomers of AP6 by fractional crystallization of thel-lysine salt ofdl-AP6. We also report the pharmacological responses of kainate / AMPA, NMDA, lateral perforant pathl-AP4 receptors and the CA1 QUIS-sensitized site tod- andl-AP6, and compare these responses to thed- andl-isomers of AP3, AP4, AP5 and AP7. Thed-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respectivel-isomers. Whilel-AP4 andl-AP5 cross-reacted with NMDA andl-AP4 receptors,l-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC50 = 40 μM). Its IC50 values for kainate / AMPA, NMDA andl-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization tol-AP6 was reversed byl-α-aminoadipate.

Evidence for a common component in kinetically distinct transport systems of Saccharomyces cerevisiae

SummaryTwo lines of evidence suggest that amino acid transport systems and the methylamine/ammonia transport system of Saccharomyces cerevisiae may share a common component or components.1.Mutant strains have been derived which are defective in transport activity for methylamine and for amino acids. A single pleiotropic mutation appears to be responsible for the observed reduction in the various transport activities.2.Transport systems for amino acids and for methylamine are sensitive to a similar degree to inhibition by proton conducting uncouplers, ATPase inhibitors, and ionophores.

Characterization of a Dio-9-resistant strain of Saccharomyces cerevisiae

Abstract The ATPase inhibitor Dio-9 effectively suppressed a number of physiological processes in a wild-type strain of Saccharomyces cerevisiae , X2180-1A. Low levels of the antibiotic inhibited cell growth, amino acid transport, hydrogen ion efflux, and ATPase activity. In addition, Dio-9 acted as a permeabilizing agent for the yeast plasma membrane. A mutant yeast strain, XC24, was selected on the basis of its ability to grow on minimal medium containing 200 μg/ml of Dio-9. Strain XC24 had acquired a pH-conditional ability to resist the permeabilizing effects of Dio-9. In addition, amino acid transport and hydrogen ion pumping exhibited a reduced senstivity to Dio-9 at low pH in the mutant strain. Strain XC24 was also resistant to the permeabilizing effects of the basic polymers protamine and deacylated chitin.

[37a] ATP: Urea amidolyase (ADP) (Candida utilis)☆

Publisher Summary This chapter describes the assay, purification, and properties of urea amidolyase. Urea serves as the sole nitrogen source for the growth of certain yeasts and unicellular green algae that contain no detectable urease activity. Such cells possess urea amidolyase, which catalyzes Mg 2+ - and K + -dependent cleavage of a mole of urea to two moles of ammonia and one mole of carbon dioxide, concomitant with the breakdown of a mole of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate. The formation of urea amidolyase is repressed by ammonia. Two types of assays are recognized for routine use. The first involves the measurement of the rate of release of 14 CO 2 from urea- 14 C and offers the advantage of convenience and high sensitivity. This assay is, particularly useful with crude extracts, because it is largely unaffected by the presence of contaminating enzymes, such as myokinase, ATPase, and 2,4-Dinitrophenylhydrazine (DPNH) oxidase. The second method is based on a coupled spectrophotometric determination of ADP using phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase. The decrease in absorbance at 340 m μ , resulting from the oxidation of DPNH, which accompanies the conversion of pyruvate to lactate, allows a continuous determination of the rate of cleavage of ATP to ADP. This method is limited to purified amidolyase preparations, because cruder fractions often contain high levels of ATPase and other interfering activities. In Tris buffers, the optimum pH for amidolyase activity (assay method 1) is 7.8–7.9.

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

Abstract α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K m for α-aminoisobutyric acid of 270 μM, a V max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH 4 + . During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

Abstract The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.

Persistent depression of synaptic responses occurs in quisqualate sensitized hippocampal slices after exposure tol-aspartate-β-hydroxamate

Exposure of slices of rat hippocampus to quisqualic acid produces an enhanced sensitivity of neurons to depolarization by other excitatory amino acid analogues, particularly amino acid phosphonates. The phosphonates may act at extracellular sites, since their depolarizing effects are rapidly reversed by washout with phosphonate-free incubation medium. We now wish to report a novel class of excitatory amino acid analogues that induce a persistent depolarization that is not reversed by washout. Exposure of quisqualate-sensitized slices of rat hippocampus to 400 microM L-aspartate-beta-hydroxamate for 8 min results in the complete depression of extracellular synaptic field potentials. This depression persists for at least 1 h after washout of the hydroxamate compound. Analogous compounds L-glutamate-gamma-hydroxamate, D-aspartate-beta-hydroxamate and the phosphonate derivative L-2-amino-3-phosphonopropanoic acid (L-AP3) induce a similar but weaker persistent depression of the field potentials. Previous studies also demonstrated that exposure of hippocampal slices to L-alpha-aminoadipate blocks or reverses quisqualate sensitization, making the neurons unresponsive to depolarization by phosphonate compounds. We now report that L-alpha-aminoadipate also blocks or reverses the persistent depolarization of quisqualate-sensitized neurons which is induced by exposure to the hydroxamates or L-AP3.

Transport and metabolism of N-δ-chloroacetyl-l-ornithine by Saccharomyces cerevisiae

Abstract The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids (M. Grenson, C. Hou, and M. Crabeel, 1970, J. Bacteriol. 103, 770–777; J. Rytka, 1975, J. Bacteriol.121, 562–570; C. Darte and M. Grenson, 1975, Biochem. Biophys. Res. Commun.67, 1028–1033). We have previously demonstrated that this transport system can be inhibited by the amino acid, N-δ-chloroacetyl- l -ornithine (NCAO) (F. S., Larimore and R.J. Roon, 1978, Biochemistry17, 431–436). In the present study radiolabeled NCAO was synthesized and its transport and metabolism studied. Under initial rate conditions: (a) NCAO was transported by the general amino acid transport system with a Km of 52 μ m , a V of 32 nmol/min/mg cells, and a pH optimum of 5.0; (b) the V for NCAO transport in gap mutants, which lack the general amino acid transport system, was approximately 1% of that observed with wild-type cells; (c) the V for NCAO in cells deprived of glucose was less than 5% of that observed when glucose was present. NCAO was transiently concentrated more than 1000-fold by yeast cells when glucose served as an energy source. The internal pool of NCAO was metabolized by the yeast cells and the products were excreted. When 100 μ m [14C]NCAO was incubated with a yeast cell suspension for 8 h, more than 95% of the compound was converted into two ninhydrin-negative excretory products. The effect of NCAO on the growth of yeast cells was determined. Wild-type strains did not grow when 1 m m NCAO was present in the medium. The growth of gap mutants was not inhibited by 1 m m NCAO.

Separation and differential sensitivity toward avidin of carbamyl phosphate synthetase and urea amidolyase

This enzyme is remarkably sensitive to inhibition by highly purified egg white avidin, a phenomenon which can be completely prevented by inclusion of excess biotin in the assay system. Inasmuch as avidin has been found to selectively inhibit all known biotinenzymes and, indeed, is now accepted as a diagnostic tool for the detection of such reactions [2], the proposal was made that UALase belongs to that class of enzymes which contain biotin in the form of a bound, functionally-active prosthetic group. A recent publication by Wellner, Santos and Meister [3], asserting that the glutamine-dependent carbamyl phosphate synthetase (CPSase, EC 2.7.2.5) of Escherichia coli may also be a biotinznzyme, has focused our attention on the relevant question of whether these two enzymic processes might possibly represent analogous activities of the same protein molecule. At this time we wish to report results which indicate that not only is each reaction catalyzed by a