Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.     

Acute metabolic effects of ammonia in mouse brain

Acute effects of intraperitoneal administration of ammonium chloride (200 mg/kg) on Na⁺,K⁺-ATPase and amino acid content of the glutamate family (glutamate, aspartate, alanine, glutamine, and GABA), as well as the enzymes involved in the metabolism of these amino acids, have been studied in the different regions of brain and liver in mice. A significant increase in the activity of Na⁺,K⁺-ATPase was observed in the cerebellum, cerebral cortex, and brain stem. A similar increase in the activity of glutamate dehydrogenase was observed in the brain stem, while a moderate increase in the activity of this enzyme was observed in the cerebral cortex and liver in the mice treated with ammonium chloride. In all three regions of brain, a 50% decrease was observed in the activity of alanine aminotransferase, while the activity of aspartate aminotransferase significantly rose in the brain stem. The activity of glutamine synthetase did not change much in the three regions of brain, and a significant fall was registered in the liver. The activity of tyrosine aminotransferase showed a rise in the cerebellum, brain stem, and in liver. Not much change was observed in the protein content in either brain or liver, whereas there was a 1.5-fold increase in the total RNA content in the liver of the animals treated with ammonium chloride. Under the experimental conditions, there was an increase only in the content of glutamine, of all the amino acids tested, in the cerebral cortex and liver. Similar results were obtained with homogenates of tissues enriched with ammonium chloride (in vitro) for the enzyme systems studied. These results are discussed, and the probable metabolic and functional significance of ammonia in brain is indicated.     

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.     

Purification and properties of pig kidney glutamate dehydrogenase.

Glutamate dehydrogenase from pig kidney has been purified to homogeneity by means of affinity chromatography on matrix bound Cibacron Blue F3G-A and gel chromatography on Sepharose 6B. The enzyme exhibits allosteric properties with the substrates alpha-ketoglutarate, ammonium, and NADH, respectively. GTP is a strong inhibitor which strengthened the cooperative interactions between the ammonium binding sites. ADP as an activator relieves the inhibition by GTP. Like glutamate dehydrogenase from bovine liver, glutamate dehydrogenase from pig kidney shows the ability of self-association, too. The sedimentation coefficient increases from 13.5 S at 0.07 mg protein/ml to 19.4 S at 1.32 mg protein/ml. In the sodium dodecylsulphate gel electrophoresis the enzyme migrates as a single band with a molecular-weight at 51000.     

Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors

A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.     

Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells.

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.     

Comparison of Enzymatic Reduction of Diphosphopyridine Nucleotide with Ammonia and Urea

In investigating the properties of the dehydrogenating system from ammonia to nitrate, it has been observed that DPN is reduced with urea also in the presence of an enzyme from fowl liver. The properties of the enzyme are very closely similar to those of ammonium dehydrogenase. However, some fundamental differences have been demonstrated by comparing these two enzymes, especially concerning reduction of TPN, substrate inhibition, formation of some nitrogenous metabolites and reoxidation of DPNH in the reaction mixture. The reaction was postulated to be a dehydrogenation of urea by an enzyme. This activity has also been demonstrated in silkworm and yeast.     

Crystallization and some properties of glutamate dehydrogenase from rat liver

1. Glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase, EC 1.4.1.3) from rat liver has been crystallized with a method carefully avoiding all denaturating agents. A 236-fold purification was achieved at a yield of 20%. The specific activity was 185 units/mg protein. The enzyme was homogeneous by analytical zone electrophoresis and sedimentation studies. The s0(20),w value was 13.2. 2. Sedimentation studies in the analytical ultracentrifuge and the behaviour of the enzyme in the disc-electrophoresis revealed that glutamate dehydrogenase from rat liver did not undergo a reversible association-dissociation reaction as reported of glutamate dehydrogenase of nearly all other mammalians. 3. Using antibodies prepared against crystalline bovine liver glutamate dehydrogenase, no immunological differences between the rat and the bovine liver enzyme could be observed.     

THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.     

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE-cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min^-1 (molecule of GDH)^-1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP-GDH is composed of six identical polypeptidic chains.
The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP-GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2-mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α-ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K_m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K_m values, indicate that the NADP-GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.     

A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase

Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

Rapid detoxification of infused ammonium by the anesthetized rat

Anaesthetized rats were given an i.v. overload of 200 mmoles of ammonium acetate. Plasma ammonium levels were not altered for up to 20 minutes after the end of the infusion. The load of ammonium, however, increased the overall non-protein nitrogen content of circulating plasma, as for the increase in urea and amino acids (alanine, phenylalanine, aspartate + asparagine and glutamate + glutamine). The activities of glutamine synthetase was found increased in liver, muscle and kidney; and glutamate dehydrogenase increased in liver and decreased in muscle and kidney. Adenylate deaminase decreased in all the studied tissues. The fast enzyme and plasma metabolite adaptations to ammonium overload were all in the sense of favoring the incorporation of ammonium into amino acids (later into urea) as well as to avoid their deamination, thus effectively removing the excess ammonium from the bloodstream.     

Immunochemical and enzymatic studies and glutamate dehydrogenase and a related mutant protein from Neurospora crassa

Roberts, D. B. (University of Cambridge, Cambridge, England). Immunochemical and enzymatic studies on glutamate dehydrogenase and a related mutant protein from Neurospora crassa. J. Bacteriol. 91:1888-1895. 1966.-When an investigation was made of the inhibition of Neurospora glutamate dehydrogenase by bivalent and univalent antibodies, it was shown that the enzyme inhibition is not complete even with excess antibodies. The residual activity was some three times greater with bivalent antibodies, in spite of the observation that the ratio of inhibiting antibodies to catalytic sites was 2:1 for both types of antibody. Substrates did not affect the inhibition of enzyme activity, nor did antibodies affect the K(m) for either substrate. An allosteric mechanism for the inhibition of glutamate dehydrogenase by antibodies is proposed. It was also demonstrated that the mutant protein am-3 can be activated, to show glutamate dehydrogenase activity, by a number of activators. The requirement for the activation was the presence of a carboxymethyl group. The data suggest that the nonactivated protein has two combining sites for l-glutamate: the catalytic and activating sites. The wild-type enzyme has only one of these sites. Because the activating site is distinct from the catalytic site, an allosteric mechanism was postulated for activation. Inhibition of am-3 activity by antibodies is achieved either by a mechanism similar to the inhibition of wild-type activity or by the antibodies preventing the activation of the mutant protein. The am-3 protein can be activated by antibodies. Consequently, there appeared to be a relation the phenomena of enzyme inhibition and am-3 activation by antibodies; i.e., they alter the configuration of the catalytic site. This alteration was necessary for the activation of am-3, but inhibited the activity of the wild-type enzyme.     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Glutamate and aspartate transport in rat brain mitochondria

1. Rat brain mitochondria did not swell in iso-osmotic solutions of ammonium or potassium (plus valinomycin) glutamate or aspartate, with or without addition of uncouplers. 2. Glutamate was able to reduce intramitochondrial NAD(P)(+); aspartate was able to cause partial re-oxidation. 3. These effects were inhibited by threo-hydroxy-aspartate in whole but not in lysed mitochondria. 4. The existence of a ;malate-aspartate shuttle' for the oxidation of extramitochondrial NADH was demonstrated. This shuttle requires the net exchange of glutamate for aspartate across the mitochondrial membrane. 5. Extramitochondrial glutamate did not inhibit intramitochondrial glutaminase under conditions in which the inhibition in lysed mitochondria was virtually complete. 6. The glutaminase activity of these mitochondria was not energy-dependent. 7. We conclude that these mitochondria do not possess a glutamate-hydroxyl antiporter similar to that of liver mitochondria nor a glutamate-glutamine antiporter similar to that of pig kidney mitochondria, but that they do possess a glutamate-aspartate antiporter.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze

Structural analysis of a cytosolic glutamine synthetase from Camellia sinensis (CsGS) has been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of GS. The disordered residues and their distribution in CsGS are in close comparison to earlier reported GS. The 3-D structure of CsGS also showed high degree of similarity with the only known crystal structure of GS from Zea mays. The K _m values observed with recombinant CsGS for all the three substrates are higher compared to rice, Arabidopsis, maize and human. This suggests lower affinity of CsGS for substrates. Further, kinetic mechanism of CsGS catalysis was investigated using initial velocity analysis and product inhibition studies. Initial velocity data eliminate the possibility of ping-pong mechanism and favor the random mechanism of catalysis. Through product inhibition studies, ADP was found to be a competitive inhibitor with respect to ATP and noncompetitive inhibitor versus both glutamate and ammonium. While, glutamine and inorganic phosphate were found to be non-competitive inhibitors of ATP, glutamate and ammonia. Taken together, these observations are consistent with a random catalysis mechanism for the CsGS where the binding order of certain substrates is kinetically preferred by the enzyme.