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In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.

1. In freshly prepared isolated rat liver cells there is a lag in gluconeogenesis from lactate. The magnitude of the lag increases with increasing lactate concentration. 2. The lag is virtually abolished by lysine. 3. A few other amino acids (tyrosine, arginine, asparagine, ornithine) and NH(4)Cl had effects similar to, but less pronounced than, lysine during the early stage of incubation. Lysine was unique in accelerating gluconeogenesis beyond the lag period. 4. The effects of the accelerators are not additive. 5. Glycine, serine, threonine, cysteine, tryptophan and histidine at 2mm markedly inhibit (>20%) gluconeogenesis from lactate. 6. Oleate, which promotes gluconeogenesis from lactate by supplying acetyl-CoA required for the pyruvate carboxylase reaction, had no effect on the lag, yet oleate oxidation showed no lag. 7. Preincubation of cells decreased the lag and decreased the magnitude of the lysine effect. 8. Pyruvate (added at 1mm to give an initial [lactate]/[pyruvate] ratio of 10) also abolished the lag and decreased the lysine effect by about 50%. 9. Lysine reversed the inhibition by ethanol of gluconeogenesis from lactate. 10. All accelerators increased the rate of re-oxidation of cytosolic NADH as shown by a rapid re-adjustment of the [lactate]/[pyruvate] ratio on addition of 10mm-lactate. 11. The accelerated rates of gluconeogenesis are associated with an increased formation of aspartate and glutamate and especially alanine. 12. The existence of the lag period can be explained on the basis of the fact that the accumulation of pyruvate during the lag diverts oxaloacetate from gluconeogenesis to malate formation, i.e. that the re-oxidation of cytosolic NADH takes precedence over gluconeogenesis. This means that much oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred twice from the mitochondria to the cytosol by the aspartate shuttle. Under these conditions the operation of the shuttle limits the rate of gluconeogenesis from lactate. Lysine and other accelerators may increase the effectiveness of the shuttle by providing components of the aspartate aminotransferases involved. The question of why lysine specifically accelerates gluconeogenesis beyond the lag period is discussed.

High activity of taurine:alpha-ketoglutarate aminotransferase was found exclusively in cell-free extracts of Achromobacter superficialis and A. polymorph. The former was chosen for characterization of the enzymatic reaction. The enzyme activity was enhanced by addition of beta-alanine to the growth medium. The product from alpha-ketoglutarate was identified as l-glutamate. Another product has been isolated, purified, and identified as sulfoacetaldehyde (2-oxoethanesulfonate), a deamination product from taurine, by comparison between the 2,4-dinitrophenylhydrazones of the synthetic and enzymatic products on the basis of studies by paper chromatography, by visible, infrared, and nuclear magnetic resonance spectrophotometries, and by elemental analysis. This enzymatic transamination was found to proceed stoichiometrically and reversibly as follows: NH(2).CH(2).CH(2).SO(3)H + HOOC.CH(2).CH(2).CO.COOH right harpoon over left harpoon OHC.CH(2).SO(3)H + HOOC.CH(2).CH(2).CH(NH(2)).COOH.

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.

The production of glutamic gamma-semialdehyde, an intermediate in the synthesis of proline, was inhibited in Escherichia coli by physiological concentrations of penicillin. Sucrose (0.6 m) and sodium chloride (0.1 m) prevented penicillin inhibition of glutamic gamma-semialdehyde synthesis. Cells which were in the stationary phase, or which had been permitted to metabolize without growth, were insensitive to the effects of penicillin on glutamic gamma-semialdehyde synthesis.

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.