Stimulation of L-asparate beta-decarboxylase formation by L-glutamate in Pseudomonas dacunhae and Improved production of L-alanine.

The formation of L-asparate beta-decarboxylase by Pseudomonas dacunhae was compared on media containing a variety of organic acids and amino acids as a carbon source. Although the enzyme was formed constitutively when the organism was grown on basal medium or on that containing tricarboxylic acid cycle intermediates, it was induced twofold by L-glutamate and repressed one-tenth by L-serine. L-Glutamine, L-proline, L-leucine, glycine, and L-threonine also showed induction effects lower than that of L-glutamate. L-Glutamate derepressed the serine effect. This glutamate effect was observed effect was observed with other microoganisms, e.g., Achromobacter pestifer and Achromobacter liquidum. Since the intermediates from L-glutamate metabolism had no effect, this induction effect was specific to L-glutamate. The formation of some glutamate-related enzymes was measured and is discussed in relation to the formation of L-asparate beta-decarboxylase. L-Asparate beta-decarboxylase was purified to an electrophoretically homogenous state from L-glutamate-grown cells of P. dacunhae, and some properties were compared with those of the enzyme from fumarate-grown cells. The two enzymes were identical in disc electrophoresis, molecular weight, and some enzymatic properties. The industrial production of L-alanine from L-aspartic acid acid was improved by using the culture broth with highly induced L-asparate beta-decarboxylase (9.4 U/ml of broth).     

Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate beta-Decarboxylase Activity and Application to l-Alanine Production

Whole cells of Pseudomonas dacunhae containing l-aspartate beta-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate beta-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45 degrees C, respectively. Immobilized P. dacunhael-aspartate beta-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM alpha-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate beta-decarboxylase activity was observed over a 31-day period.     

Enzymatic Production of l-Alanine by Pseudomonas dacunhae

To establish an advantageous method for the production of l-alanine, a procedure was studied for converting l-aspartic acid to l-alanine by microbial l-aspartic beta-decarboxylase. A number of organisms were screened to test their ability to form and accumulate alanine from aspartic acid. Pseudomonas dacunhae was selected as the most advantageous organism. With this organism, enzyme activity as high as 3,910 muliters of CO(2) per hr per ml of medium could be produced by shaking the culture at 30 C in the medium containing ammonium fumarate, sodium fumarate, corn steep liquor, peptone, and inorganic salts. For the enzymatic conversion of l-aspartic acid to l-alanine, the culture broth was employed as the enzyme source. A large amount of l-aspartic acid (as much as 40% of the broth) was converted stoichiometrically to alanine in 72 hr at 37 C. Furthermore, appropriate addition of a surface-active agent to the reaction mixture was found to be highly effective in shortening the time required for the conversion. Accumulated l-alanine was readily isolated in pure form by ordinary procedures with ion-exchange resins. Yields of isolated l-alanine of over 90% from l-aspartic acid were easily attainable.     

Recirculating bioreactor-separator system for simultaneous biotransformation and recovery of product: Immobilized L-aspartate beta-decarboxylase reactor system

A new combined bioreactor-separator system was designed and its operational feasibility demonstrated in order to develop a bioprocess that enables us to handle simultaneous biotransformation and recovery of product by crystallization. Enzymatic conversion of L-aspartate to L-alanine by L-aspartate beta-decarboxylase from Pseudomonas dacunhae (ATCC 21192) was used as a model system for this study to demonstrate the principles involved in the bioprocess design. Immobilized cells of P. dacunhae containing the enzyme were fluidized in a tapered column type of bioreactor and a filter-crystallizer combination was used as a separator unit in our experimental system.It was found that almost a theoretical yield was achieved, and the process control for both the bioreactor operation and separation was relatively easy. The Production systems, namely, the recirculating bioreactor separator combination system and the conventional batch reactor system, were analyzed and compared based on the results obtained form this study, and it was found that a significant cost reduction, by about 20%, can be achieved when the recirculating bioreactor-separator combination system was employed. Based on these findings, it is anticipated that the conceptual design of the bioreactor-separator combination system evaluated in this study has some potential for industrial application.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Regulation of the expression of plasmid determination responsible for caprolactam degradation by bacteria of the genus Pseudomonas]

On the basis of the study of some Tn5 induced mutants in Pseudomonas putida strain BS836 containing the plasmid pBS268 coding caprolactam degradation, growth on caprolactam and its intermediates, and the data on the induction of oxidative activities in plasmid containing P. putida strain BS831 it was shown that plasmid and chromosome genes regulated the expression of CAP-determinants. The regulation has some elements of the negative control mechanism. Caprolactam is the inducer of the synthesis of key enzymes cleaving it and its intermediates (aminocaproic and adipic acids). At the same time each of its intermediates induced the synthesis of enzymes responsible for its cleavage.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus.

Two bacteriolytic enzymes secreted by Achromobacter lyticus M497-1 were purified and identified as being very similar (considering their amino acid composition and N-terminal sequence) to alpha- and beta-lytic proteases from Lysobacter enzymogenes. A 1.8-kb EcoRI fragment containing the structural gene for beta-lytic protease was cloned from A. lyticus chromosomal DNA. The protein sequence deduced from the nucleotide sequence was identical to the known sequence of beta-lytic protease, except for six residues. The nucleotide sequence revealed that the mature enzyme is composed of 179 amino acid residues with an additional 195 amino acids at the amino-terminal end of the enzyme, which includes the signal peptide, thus indicating that the enzyme is synthesized as a precursor protein.     

The microbial degradation of cyclohexanecarboxylic acid by a beta-oxidation pathway with simultaneous induction to the utilization of benzoate

The metabolism of cyclohexanecarboxylic acid by a bacterium, designated PRL W19, follows a pathway involving beta-oxidation of coenzyme A intermediates analogous to the classical oxidation of fatty acids. The organism appears to have the property for the constitutive metabolism of caproic acid, and cell extracts contain high levels of the enzymes required for the functioning of the fatty acid cycle. However, the metabolism of cyclohexanecarboxylic acid requires induction by growth or incubation with an appropriate substrate. Extracts of induced cells contain several enzyme activities which are synthesized in response to the induction process. These enzymes include cyclohexanecarboxyl-CoA synthetase, cyclohexanecarboxyl-CoA dehydrogenase, 1-cyclohexenecarboxyl-CoA hydratase, and trans-2-hydroxycyclohexanecarboxyl-CoA dehydrogenase. A characteristics feature of this organism is that it becomes induced for the metabolism of benzoate and catechol during growth on cyclohexanecarboxylic acid, but benzoate does not appear to be an obligatory intermediate in the metabolism of cyclohexanecarboxylic acid.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium.

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : IV. Induction of Vanillic Acid Formation

Kinetin is used as an elicitor to induce vanillic acid formation in cell suspension cultures of Vanilla planifolia. Maximal induction is observed at a kinetin concentration of 20 micrograms per gram of fresh weight of cells. Vanillic acid synthesis is observed a few hours after elicitation. The effects of kinetin on the activity of some enzymes of the phenylpropanoid pathway, i.e. phenylalanine ammonia-lyase, 4-hydroxycinnamate:coenzyme A ligase and uridine 5′-diphosphate-glucose:trans-cinnamic acid glucosyltransferase, are reported and compared to the effects of chitosan. The former two enzymes are induced by chitosan with a maximum activity of approximately 25 to 40 hours after elicitation. All three enzymes are induced by kinetin with maximum activities for phenylalanine ammonia lyase and 4-hydroxycinnamate:coenzyme A ligase at approximately 50 hours after induction, whereas maximum glucosyltransferase activity is seen already after 24 hours. Furthermore, both elicitors induced the formation of lignin-like material, whereas only kinetin induced vanillic acid biosynthesis. Finally, kinetin but not chitosan induces catechol-4-O-methyltransferase activity, catalyzing the formation of 4-methoxycinnamic acids, which were shown to be intermediates of hydroxybenzoic acid biosynthesis within cells of V. planifolia. It is suggested that this methyltransferase is directly involved in the biosynthesis of vanillic acid.     

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.     

Properties of an L-glutamate-induced acid tolerance response which involves the functioning of extracellular induction components

Escherichia coli became more acid tolerant following incubation for 60 min in a medium containing L-glutamate at pH 7.0, 7.5 or 8.5. Several agents, including cAMP, NaCl, sucrose, SDS and DOC, prevented tolerance appearing if present with L-glutamate. Lesions in cysB, hns, fur, himA and relA, which frequently affect pH responses, failed to prevent L-glutamate-induced acid tolerance but a lesion in L-glutamate decarboxylase abolished the response. Induction of acid tolerance by L-glutamate was associated with the accumulation in the growth medium of a protein (or proteins) which was able to convert pH 7.0-grown cultures to acid tolerance, and the original L-glutamate-induced tolerance response was dependent on this component(s). Acid tolerance was also induced by L-aspartate at pH 7.0 and induction of such tolerance was dependent on an extracellular protein (or proteins). The L-glutamate and L-aspartate acid tolerance induction processes are further examples of a number of stress tolerance responses which differ from most inductions in that extracellular components, including extracellular sensors, are required.     

Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

Effects of amino-acid composition of the medium and addition of fatty-acid derivatives on the induction of lipogenic enzymes in cultured hepatocytes

Effects of essential and non-essential amino acids on induction of lipogenic enzymes were investigated in cultured rat hepatocytes. Glucose-6-phosphate dehydrogenase was markedly induced by the addition of essential amino acids alone to the cultured medium, but was not induced by non-essential amino acids. Fatty-acid synthetase was also markedly induced by a combination of both amino-acid types (more than by either type of amino acid alone). However, acetyl-CoA carboxylase and malic enzyme were slightly induced by the addition of essential and/or non-essential amino acids. When various kinds of fatty acids were individually added to the medium, the lipid-dependent decreases in lipogenic enzyme inductions were in the following order: 18:2 greater than 20:4 greater than 18:1 greater than 16:0. When either linoleic acid, linoleoyl-CoA or trilinolein was added to the medium, linoleic acid was more effective as an inhibitor of the induction, without impairing the viability of cells.     

Pre-steady-state measurement of intrinsic secondary tritium isotope effects associated with the homolysis of adenosylcobalamin and the formation of 5'-deoxyadensosine in glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze a variety of reactions that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. For all the enzymes that have been examined, the homolysis step is kinetically indistinguishable from abstraction of hydrogen from the substrate (or protein), implying that deoxyadenosyl radical is formed only as a fleeting intermediate. To examine how these two steps are coupled together, we have used pre-steady-state, rapid quench techniques to measure the alpha-secondary tritium isotope effect associated with the formation of 5'-deoxyadenosine when the enzyme is reacted with [5'-(3)H]-adenosylcobalamin and L-glutamate. Surprisingly, a large inverse equilibrium isotope effect of 0.72 +/- 0.04 was found for the overall reaction, indicating that the 5'-C-H bonds become significantly stiffer on going from adenosylcobalamin to 5'-deoxyadenosine, even though the 5'-carbon remains formally sp(3) hybridized. The kinetic isotope effect for the formation of 5'-deoxyadenosine was 0.76 +/- 0.02, which suggests a late transition state for the reaction.     

Extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6: preparation of it and its properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain, Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6-1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase from Streptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1-0.5% promoted the secretion of the extracellular glutamate oxidase.     

Observations on the bacterial oxidation of chlorophenoxyacetic acids

Summary Three bacterial strains, one ofF. peregrinum (Stapp and Spicher) and two Achromobacter strains, have been isolated from soil and shown to decompose either 2,4-D, MCPA orp-chlorophenoxyacetic acid. Aerobic conditions are essential for the bacterial decomposition of 2,4-D. Pretreatment of soil with one of the three chlorophenoxyacetic acids accelerated the rate of breakdown of either of the other two. In a liquid medium, growth of theF. peregrinum strain caused breakdown of 2,4-D and liberated 76% of the chlorine in 2,4-D in ionic form. An unknown acidic substance, colourless in acid solution but forming a yellow sodium salt has been detected in cultures ofF. peregrinum or an MCPA-decomposing Achromobacter strain growing inp-chlorophenoxyacetate medium. The bacterial oxidation of chlorophenoxyacetic acid herbicides was attributed to adaptive enzyme formation. Respiration experiments showed that the oxidation of 2,4-D or ofp-chlorophenoxyacetic acid is incomplete. 4-Chloro-2-hydroxyphenoxyacetic acid and 4-chlorocatechol may be metabolic intermediates in the case ofp-chlorophenoxyacetic acid, but no intermediary metabolites have as yet been established for 2,4-D.     

Excitatory Amino Acid-Induced Formation of Inositol Phosphates in Guinea-Pig Cerebral Cortical Slices: Involvement of Ionotropic or Metabotropic Receptors?

In cerebral cortical slices from the guinea-pig, quinoxalinedione derivatives antagonised the generation of 3H-inositol phosphates evoked by the excitatory amino acids quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid but were without effect on the trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid and L-glutamate responses. Omission of calcium from the medium reduced the accumulation of 3H-inositol phosphates induced by incubation with trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (incubation for 45 min) by greater than 50%, whereas the responses to L-glutamate and the two other amino acid analogues were reduced by approximately 20%. Generation of inositol 1,4,5-trisphosphate over a 30-s period by treatment with quisqualate, trans-DL-1-amino-1,3-cyclopentane-dicarboxylic acid, KCl, and carbachol was abolished in the presence of nominally calcium-free medium. L-Glutamate induced a large, rapid increase in inositol 1,4,5-trisphosphate mass (more than three-fold), which was, however, unaffected by omission of calcium from the medium. These results indicate that of the excitatory amino acids tested, only L-glutamate may be classed as a metabotropic receptor agonist in guinea-pig cerebral cortical slices with respect to generation of inositol phosphates. The other agents appear to stimulate accumulation of inositol phosphates, at least in part through some mechanism requiring the presence of extracellular Ca2+, presumably Ca2+ entry.