Allen T. Phillips

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H/sup +/) are used to separate the product, (/sup 14/C)glutamine, from unreacted (U-/sup 14/C)glutamate and other labeled compounds, particularly ..gamma..-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measureglutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.

Glucocorticoid stimulation of glutamine synthetase production in cultured rat glioma cells.

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C‐6 glioma cultures. The Km values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM‐ Mg2+ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM‐L‐methionhe sulfoximine. The addition of 1 μM‐cortisol to C‐6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96‐h period, while dexamethasone at the same concentration elevated the level some 7‐10‐fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.

Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid

Abstract1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 μM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 μM IAA.

L-GLUTAMIC ACID DECARBOXYLASE IN NON-NEURAL TISSUES OF THE MOUSE

Abstract— Low levels of γ‐aminobutyric acid (GABA) and of glutamic acid decarboxylase (GAD) activity have been detected in mouse kidney, liver, spleen and pancreas. Quantitation of both 14CO2 and [14C]GABA produced in radiometric assays from [U‐14CJglutamic acid has shown that measurement of 14CO2 evolution alone is not, in all cases, a valid estimate of true GAD activity. As evidenced by increased ,14CO2 production upon addition of NAD and CoA to assay mixtures, radiometric assay of GAD activity in crude homogenates may yield 14CO2 via the coupled reactions of glutamic acid dehydrogenase and a‐ketoglutarate dehydrogenase. The addition of 1 mM aminooxyacetic acid (AOAA) to assays of kidney homogenates inhibited [,14C]GABA production 92 per cent while 14CO2 production was inhibited only 53 per cent. No evidence was found to confirm the reported existence of a second form of the enzyme, GAD II. previously described by Haber el al. (Haber B., Kuriyama K. & Roberts E. (1970) Biochem. Pharmac. 19, 1119‐1136). Based on sensitivity‐to AOAA and chloride inhibition, the GAD activity in mouse kidney is. apparently, indistinguishable from that of neural origin.

Identification of two new members of the l-aminocyclopropane-l-carboxylate synthase-encoding multigene family in mung bean

The key enzyme regulating ethylene biosynthesis in higher plants is 1-aminocyclopropane-1-carboxylate (ACC) synthase. In mung bean (MB), the existence of three genes encoding this enzyme has previously been reported [Botella et al., Plant Mol. Biol. 18 (1992) 793-797], one of which corresponds to a full-length indole-3-acetic acid-inducible cDNA [Botella et al., Plant Mol. Biol. (1992) 425-436]. In this paper we report the cloning of two new genomic sequences coding for ACC synthase in MB (MAC-4 and MAC-5). MAC-4 is 1340 bp long and encodes 388 amino acids (aa) while MAC-5 is 1393 bp long and encodes for 391 aa. Genomic Southern analysis suggests the existence of only one copy of each gene in the genome.

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments.

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.

Intracellular amino acid content of neuronal, glial, and non-neural cell cultures : the relationship to glutamic acid compartmentation

Abstract— A protocol for the accurate determination of intracellular levels of amino acids in tissue cultured cells has been developed and used in the measurement of intracellular amino acids levels in neuronal, glial, and non‐neural cell lines, with the objective of establishing morphological correlates for large and small glutamic acid compartments and of examining hypotheses for the morphological basis of glutamic acid compartmentation. This survey of intracellular amino acid levels has revealed striking differences among the cell lines tested, but these differences did not correlate with cell type, i.e. neuronal vs glial, in contrast to earlier results (Rose, 1968) based on bulk separated neuronal and pial fractions from rat brain. Amino acid levels were found to be dependent upon tissue culture conditions, yet reproducible differences could be observed when growth and experimental conditions were carefully controlled. Glutamic acid levels for various cell lines ranged from 50.8 ± 14.3 to 158 ± 8.5 nmol/mg protein. Intracellular glutamine levels demonstrated even greater difference, with values ranging from 0.8 ± 0.2 to 107 ± 42.4 nmol/mg protein. Statistically significant differences in intracellular amino acid levels between cell lines were also observed for aspartic acid, praline, glycine, alanine, valine, cystathionine, isoleucine, and leucine. A number of cell lines demonstrating highly elevated elevated levels of γ‐aminobutyrate and β‐alanine were identified. The significance of neuronal and glial levels of glutamic acid, glutamine and γ‐aminobutyrate to models for glutamic acid compartmentation is discussed.

A stable oligomer of Bacillus thuringiensis delta-endotoxin, CryIIIA

Abstract A purified sample of crystalline delta-endotoxin, CryIIIA, formed a stable oligomer in various gel systems and in solution. Non-heated samples of alkaline-solubilized CryIIIA demonstrated a high MW band with Laemmli buffer/SDS-PAGE. Furthermore, CryIIIA produced a single band with Native-PAGE migrating between BSA dimer (132,000 Da) and s-amylase (200,000 Da) standards. A related CryIII toxin, CryIIIB2, demonstrated a similar size band on the same Native-PAGE system, suggesting a general phenomenon of CryIII oligomerization. When 125 I-CryIIIA was solubilized in 0.050 M potassium phosphate, pH 7.0, and run as a non-heated sample in neutral potassium phosphate buffer SDS-PAGE, a high MW band was detected, indicating that the oligomer could theoretically form under less alkaline conditions such as would be encountered in a coleopteran midgut. Analytical ultracentrifugation of alkaline-solubilized CryIIIA provided strong evidence for a stable species in solution with a MW estimate of 117,200, 113,300 or 101,500 ± 4800 Da for three separate sedimentation equilibrium experiments. These data demonstrate that CryIIIA most likely exists as a dimer in solution and support the premise that delta-endotoxins from oligomeric structures which may be crucial for sustaining large conductance ion channels in host brush border membranes.

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyls.

1-Aminocyclopropane-1-carboxylate (ACC) synthase, EC 4.4.1.14, was purified to homogeneity from etiolated mung bean hypocotyl segments. This was made possible by the ability to elevate the enzyme level markedly through hormone treatments and by stabilization of the enzyme with high phosphate concentrations. The four-step procedure resulted in 1050-fold purification with 25% yield, and consisted of stepwise elution from hydroxylapatite, chromatography on phenyl-Sepharose CL-4B, gradient elution from hydroxylapatite, and fast protein liquid chromatography (FPLC) on a MonoQ anion-exchange column. FPLC-purified ACC synthase migrated as a single band of Mr 65,000 on denaturing polyacrylamide gel electrophoresis. The molecular weight of native enzyme by Bio-Gel A-0.5 M chromatography was 125,000, indicating that the enzyme probably exists as a dimer of identical 65,000 Mr subunits. The mung bean ACC synthase exhibited a pH optimum of 8.0 for activity and a Km for S-adenosylmethionine (AdoMet) of 55 microM at 30 degrees C. It exhibited an Arrhenius activation energy of 12 kcal mol-1 degree-1 and was inactivated at temperatures in excess of 40 degrees C. The specific activity for pure ACC synthase was 21 mumol of ACC formed/mg protein/h when determined under optimal conditions with 400 microM AdoMet.

Transport of Glucose by Bifidobacterium animalis subsp. lactis Occurs via Facilitated Diffusion

ABSTRACT Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of d-[U-14C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-β-glucoside than methyl-α-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for Kt and Vmax were 14.8 ± 3.4 mM d-glucose and 0.13 ± 0.03 μmol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organisms glucose uptake.