Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica.

Glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from Anabaena cylindrica was inhibited by alanine, glycine, serine and aspartate. The effects of alanine and serine were uncompetitive with respect to glutamate, while those of glycine and asparatate were uncompetitive with respect to glutamate, while those of glycine and aspartate were non-competitive and mixed type respectively. Different pairs of amino acids and their various combinations caused a cumulative inhibition of the enzyme activity. Glutamine synthetase was also inhibited by ADP and AMP and both nucleotides affected the enzyme competitively with respect to ATP and non-competitively for glutamate. Inorganic pyrophosphate, between 2 and 3 mM, produced a very pronounced inhibiton of enzyme activity. The inhibition by PPi was uncompetitive for ATP. Various combinations of the adenine nucleotides, PPi and Pi exerted a cumulative inhibitory effect on the enzyme activity, as did the amino acids, in different combinations with either adenine nucleotides, PPi or Pi. The effects of the adenine nucleotides and the amino acids were more pronounced at higher concentrations of ammonia. Except for serine similar responses of these effectors were obtained with increasing concentrations of Mg2+. It is proposed that changes in the free concentrations of Mg2+ are important in energy-dependent regulation of the enzyme activity in this alga.     

Some properties of glutamine synthetase from the nitrifying bacterium Nitrosomonas europaea

Nitrosomonas europaea oxidizes ammonia to nitrite, thereby deriving energy for growth. Glutamate dehydrogenase (NADP+) (EC 1.4.1.4) is the main route for the incorporation of ammonia into glutamic acid, because glutamate synthase (NADPH)(EC 1.4.1.13) was not detected in cell-free extracts of N. europaea. Some properties of a partially purified glutamine synthetase (EC 6.3.1.2) have been determined, namely the effects of pH and metal ions, substrate requirements, Km and Ki values, based on biosynthetic and gamma-glutamyltransferase (EC 2.3.2.2) assays. The molecular weight of the enzyme preparation was approximately 440 000. The gamma-glutamyltransferase activity was markedly inhibited by alanine, lysine, glutamic acid, aspartic acid and serine and to a lesser extent by glycine, asparagine, arginine and histidine. Except for tryptophan and cystine, the gamma-glutamyltransferase activity was inhibited to a greater extent by these amino acids than was the biosynthetic activity. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of enzyme activity determined by either method. Of the various nucleotides tested, the gamma-glutamlytransferase activity of the enzyme was inhibited to a greater extent by di- and triphosphate nucleotides--IDP, CDP, UDP, ITP, CTP, TTP and ATP (except GDP and GTP) than by monophosphate nucleotides except AMP. Saturating concentrations of pyruvate, oxalate, oxaloacetate and alpha-ketoglutarate depressed enzyme activity. Various combinations of amino acids with adenine nucleotides exerted cumulative inhibitory effects on the transferase activity.     

Regulation of glutamine synthetase in the blue-green alga Anabaena flos-aquae

Glutamine synthetase (GS) was isolated from log phase cells and purified to a single protein as evidenced by gel electrophoresis. Protamine and ammonium sulfate precipitation and chromatography on DEAE-cellulose and Bio-Gel resulted in 380-fold purification. The enzyme was most sensitive to alanine (85% inhibition at 0.1 mM) but was also inhibited by glycine, arginine and serine. Combinations of inhibitory amino acids or nucleotides (AMP, ADP, ATP) exhibited cumulative inhibition. Cooperative inhibition was noted with CTP and any single nucleotide. Inhibition by CTP alone was uncompetitive with respect to glutamine. The enzyme was also regulated by the energy charge of the cell.     

Quantitative changes in aminoacylation of transfer RNA and in free amino acids during fructose-induced depletion of adenine nucleotides in rat liver

Fructose induces depletion of adenine nucleotides in liver and also strongly inhibits incorporation of radioactive amino acids into protein (Mäenpää, P.H., Raivio, K.O. and Kekomäki, M.P. (1968) Science 161, 1253–1254). In this study we have investigated the effects of fructose on aminoacylation of tRNA and on free amino acids in rat liver. 30 min after d-fructose (30 mmol/kg) was injected intraperitoneally into rats, liver ATP was reduced by 58%, ADP by 42%, AMP by 13%, the ATP/ADP ratio by 30%, and total adenine nucleotides by 48%. Using gas chromatography, the aminoacylation of tRNA was determined by quantifying the endogenous amino acids attached to tRNA in vivo. Aminoacylation was reduced by 31%. With different amino acids, reduction varied from 4% (asparagine plus aspartic acid) to 58% (arginine). On the other hand, the amount of free amino acids in the liver was increased by 24%. The most marked individual change was in alanine, which increased 5.7-times. This may have resulted from a combination of effects involving an increased production of alanine in muscle and liver and decreased hepatic gluconeogenesis from alanine caused by the ATP depletion.     

Inhibition of ATP phosphoribosyltransferase by AMP and ADP in the absence and presence of histidine.

AMP and ADP are linear competitive inhibitors of ATP phosphoribosyltransferase with respect to both substrates in the histidine biosynthetic direction reaction. This apparent contradiction to the proposed steady-state ordered Bi-Bi kinetic mechanism is reconciled to it on the basis of near equilibrium binding of the first substrate ATP. In the presence of histidine, AMP and ADP become positively cooperative and much more potent inhibitors of the enzyme, with AMP becoming the better inhibitor. This synergism is specific since other amino acids or nucleotides will not substitute. It is concluded, in agreement with previous workers, that energy charge could be a quantitatively important regulator of histidine biosynthesis at the metabolite level.     

Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.     

The effects of adenine nucleotides on NADH binding to mitochondrial malate dehydrogenase

The effects of adenine nucleotides on initial velocity and NADH binding have been studied with the malate dehydrogenase reaction. ATP, ADP, and AMP were inhibitors competitive with NADH and uncompetitive with oxaloacetate but caused only 50–60% inhibition at saturating concentrations. Direct fluorescence titrations indicated that saturating concentrations of the adenine nucleotides displaced 50–60% of the bound NADH from enzyme-NADH complex. Adenine and adenosine had no inhibitory effect but ADP-ribose caused complete inhibition and NADH dissociation. The possible mechanistic basis for these results and their physiological implications are discussed.     

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.     

Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography

A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.     

Isolation and Characterization of Glycine Hydroxamate-resistant Cell Lines of Nicotiana tabacum

Seven lines of haploid Nicotiana tabacum tissue culture selected for resistance to normally toxic levels of the glycine analog glycine hydroxamate, a competitive inhibitor of the glycine decarboxylase reaction, were investigated. The presence of glycine hydroxamate greatly increased the intracellular concentration of both glycine and alanine in wild type and resistant cell lines, suggesting that the inhibitor blocks both glycine- and alanine-utilizing reactions. All the resistant cell lines, whether grown in the presence or absence of glycine hydroxamate, had high intracellular concentrations of the 12 free amino acids which were analyzed, including glycine and serine. (These lines averaged 3.6 times the total amino acid content of wild-type cells in the absence of the inhibitor). The resistant cell lines were indistinguishable from wild-type cell lines in their metabolism of radioactively labeled glycine hydroxamate and glycine. Comparison of the metabolism of radioactively labeled alanine, glycolate, and glyoxylate in wild-type and α resistant line also revealed no distinctive differences. Glycine decarboxylase activities were unaltered in the resistant cell lines. The cellular toxicity of glycine hydroxamate is considered in relation to (1) the competitive inhibition by glycine hydroxamate of the glycine- and alanine-utilizing enzymes and (2) the resultant imbalances caused by high intracellular concentrations of these amino acids. The significance of elevation of total free amino acid concentration in effecting resistance to the inhibitor is discussed.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

Effect of sound and Lenzites-decayed wood on the amino acid composition of Reticulitermes flavipes

In hydrolysates of the eastern subterranean termite, Reticulitermes flavipes, the most abundant protein amino acids (μmoles) were glycine, alanine, and glutamic acid; the least abundant were methionine and histidine. Sawdust from both sound and Lenzites trabea-decayed sapwood blocks of sugar maple, loblolly pine, and slash pine was force-fed to termites. A diet of decayed rather than sound wood had little effect on protein amino acid composition of the termites; glycine content varied the most. In contrast, diet affected the free amino acid composition. Except for glutamic acid, the major protein amino acids of the termites were not the predominant free amino acids. Tyrosine and histidine were relatively more abundant as free than as protein amino acids. Greatest differences in protein amino acid compositions of sound and decayed wood were in contents of glycine, leucine, lysine, and arginine.     

Pea leaf glutamine synthetase: regulatory properties

Of a variety of purine and pyrimidine nucleotides tested, only ADP and 5'AMP significantly inhibited the Mg(2+)-dependent activity of pea leaf glutamine synthetase. They were less effective inhibitors where Mn(2+) replaced Mg(2+). They were competitive inhibitors with respect to ATP, with inhibition constant (Ki) values of 1.2 and 1.8 mm, respectively. The energy charge significantly affects the activity of glutamine synthetase, especially with Mg(2+). Of a variety of amino acids tested, l-histidine and l-ornithine were the most inhibitory, but significant inhibition was seen only where Mn(2+) was present. Both amino acids appeared to compete with l-glutamate, and the Ki values were 1.9 mm for l-histidine (pH 6.2) and 7.8 mm for l-ornithine (pH 6.2). l-Alanine, glycine, and l-serine caused slight inhibition (Mn(2+)-dependent activity) and were not competitive with ATP or l-glutamate.Carbamyl phosphate was an effective inhibitor only when Mn(2+) was present, and did not compete with substrates. Inorganic phosphate and pyrophosphate caused significant inhibition of the Mg(2+)-dependent activity.     

Relation between glutamate and adenine nucleotide levels of heart mitochondria during hypoxia

The effect of acute respiratory hypoxia in rats on mitochondrial respiration, adenine nucleotides and some amino acids of the heart was studied. The decrease in the total (ATP + ADP + AMP) and exchangeable (ATP + ADP) adenine nucleotide pool of the mitochondria was accompanied by a pronounced loss of state 3 respiration with glutamate plus malate and a slight decrease with succinate plus rothenone. The uncoupled respiration of mitochondria with glutamate and malate was decreased in the same degree as in the absence of 2,4-dinitrophenol. State 4 respiration with substrates of both types was unaffected by hypoxia. These data point to a hypoxia-induced impairment of complex I of the respiratory chain. The decrease of tissue and mitochondrial glutamate was accompanied by the elevation of alanine content in the heart and an increase in intramitochondrial aspartate. The ADP-stimulated respiration of mitochondria was correlated with mitochondrial glutamate and ATP as well as with exchangeable adenine nucleotide pools during hypoxia. The experimental results suggest that mitochondrial dysfunction induced by hypoxia may also be attributed to the low level of mitochondrial glutamate.     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. II. Effects of adenine nucleotides

• 1.1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations.
• 2.2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor.
• 3.3. Mg²⁺ decreases the inhibition produced by AMP and ADP by enhancing their I_0.5 and completely annulates the inhibitory effect of ATP.
• 4.4. In the presence of Mn²⁺ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.

     

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.