Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site

High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.     

Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin

Relative to the same site in oncomodulin, the CD ion-binding domain of rat parvalbumin exhibits much greater affinity for Ca2+ and Mg2+. As part of an effort to understand the structural basis for these differences, site-specific variants of oncomodulin have been prepared in which the amino acid residues at positions 52, 54, 57, 59, and 60 have been replaced with the residues present at the corresponding positions in rat parvalbumin. The proteins resulting from the single-site substitutions at residues 52, 54, and 57 are indistinguishable from the wild-type protein on the basis Eu3+ luminescence spectroscopy, and none of the three variants displays increased affinity for Ca2+. By contrast, the substitutions at residues 59 and 60 perturb both the Eu3+ luminescence parameters and the Ca2+ and Mg2+ affinities, and these differences are amplified when both replacements are simultaneously incorporated into the protein. The Eu3+ 7F0----5D0 spectrum of the double variant (D59E/G60E) at pH 5.0, with a maximum at 5796 A and pronounced shoulder at 5791 A, strongly resembles that obtained with pike parvalbumin. Consistent with this increased parvalbumin-like character, KCa is decreased from 0.78 microM (for the wild-type protein) to 0.41 microM, and KMg is decreased from 3.5 to 0.74 mM. Nevertheless, the affinity of the CD ion-binding domain in D59E/G60E for Ca2+ remains almost 2 orders of magnitude lower than the corresponding site in rat parvalbumin, strongly suggesting that residues besides those present in the binding loop are involved in dictating the metal ion-binding properties of the oncomodulin CD site.     

Synthesis of glutamate and aspartate in rat brain synaptosomes

ATP and glutamine are the sources of endogenous ammonia in rat brain synaptosomes. The amount of endogenous ammonia formed from exogenous ATP is not sufficient to assure the maximum rate of aspartate and glutamate accumulation in the synaptosomes utilizing pyruvate + malate. Addition of exogenous NH4+ or depolarization of synaptosome plasma membranes with high K+ concentration led to a twofold increase in the rate of accumulation of these amino acids. This indicates that both exogenous and endogenous NH4+ is involved in the synthesis of aspartate and glutamate in nerve terminals. Accumulation of glutamate was stimulated by aminooxyacetate and inhibited by haloperidol which indicates that NH4+ is bound in the reaction catalysed by glutamate dehydrogenase. Endogenous oxaloacetate derived from pyruvate metabolism was the substrate for synthesis of aspartate. Additive inhibition of aspartate accumulation by fluorocitrate and (-) hydroxyacetate shows that, in addition to the tricarboxylic acid cycle, the reaction catalysed by ATP-citrate lyase serves in the synaptosomes as another source of oxaloacetate.     

Lanthanide-binding properties of rat oncomodulin

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.     

Transport of glutamate and aspartate across the membranes of rat liver mitochondria

Chromatographic analyses have indicated that aspartate and glutamate constitute from 50–70% of the total free amino acids in freshly isolated mitochondria. Radioactive tracer studies indicate that while the l-isomers of glutamate and aspartate are rapidly accumulated by mitochondria, the d-isomers of these amino acids do not penetrate the mitochondrial membrane. The action of two inhibitory compounds, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) and tannic acid, on the transport of l-glutamate and l-aspartate has been examined. A marked inhibition of l-glutamate transfer by 1-fluoro-2,4-dinitrobenzene is observed. A corresponding effect on the transport of either l-aspartate or the anionic substrate, succinate has not been found. Tannic acid, an agent previously known to inhibit certain carrier-mediated solute fluxes in mitochondria, is shown also to inhibit the uptake of both l-glutamate and l-aspartate. These findings are consistent with the view that the mitochondrial membranes of rat liver cells contain distinct, stereospecific transport mechanisms for aspartate and glutamate.     

Analogue of aspartate and glutamate active at synapses are attractants forEscherichia coli

Summary 1. This research was carried out to compareEscherichia coli bacteria with animals in their response tol-aspartate andl-glutamate and their analogues.2. Various analogues of aspartate and glutamate known to be neurotransmitters at synapses were shown to be attractants forE. coli.3. The amino acid sequences of the animal receptors and the bacterial receptor, however, have no detectable relationship. Based on the amino acid sequence, evolutionarily the two systems appear not to be related.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Structure-activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A.

A series of analogues of neurokinin A(4-10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), all compounds except [Asp5]NKA(4-10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4-10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4-10) is in fact neurokinin B(4-10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4-10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4-10). However, [Glu5]NKA(4-10) and [Gln5]NKA(4-10) were no different from NKA(4-10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Uptake and metabolism of glutamate and aspartate by astroglial and neuronal preparations of rat cerebellum

Astrocytes, neuronal perikarya and synaptosomes were prepared from rat cerebellum. Kinetics of high and low affinity uptake systems of glutamate and aspartate, nominal rates of¹⁴CO₂ production from [U–¹⁴C]glutamate, [U–¹⁴C]aspartate and [1–¹⁴C]glutamate and activities of enzymes of glutamate metabolism were studied in these preparations. The rate of uptake and the nomial rate of production of¹⁴CO₂ from these amino acids was higher in the astroglia than neuronal perikarya and synaptosomes. Activities of glutamine synthetase and glutamate dehydrogenase were higher in astrocytes than in neuronal perikarya and synaptosomes. Activities of glutaminase and glutamic acid decarboxylase were observed to be highest in neuronal perikarya and synaptosomes respectively. These results are in agreement with the postulates of theory of metabolic compartmentation of glutamate while others (presence of glutaminase in astrocytes and glutamine synthetase in synaptosomes) are not. Results of this study also indicated that (i) at high extracellular concentrations, glutamate/aspartate uptake may be predominantly into astrocytes while at low extracellular concentrations, it would be into neurons (ii) production of -ketoglutarate from glutamate is chiefly by way of transamination but not by oxidative deamination in these three preparations and (iii) there are topographical differences glutamate metabolism within the neurons.     

The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain

1. The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain. 2. Glutamate decarboxylase is localized in the synaptosome fraction. The mean density of the particles containing the enzyme is slightly greater than those derived from cholinergic neurones, though overlap is substantial. 3. The enzyme is readily released from synaptosomes by hypo-osmotic treatment, but in the presence of Ca(2+), Na(+) and K(+) it sediments with particulate material. 4. The release and binding of the enzyme to membrane fractions by Ca(2+) were investigated. 5. Aspartate transaminase is present in brain as two isoenzymes with different kinetic properties. One isoenzyme is associated with the cytoplasm and the other with mitochondria.     

Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein.

Ninety percent of variant medium-chain acyl-CoA dehydrogenase (MCAD) alleles in patients with MCAD deficiency carry a 985 A-->G transition which causes glutamate substitution for lysine 329 in precursor (p) MCAD (K-304 in mature MCAD). We have used site-directed mutagenesis to produce three variant cDNAs encoding variant pMCAD with glutamate (Kp329E2), aspartate (Kp329D), or arginine (Kp329R) substitution for Kp329. We carried out in vitro expression of cDNAs, and incubated the translation products with isolated rat liver mitochondria. Kp329E was imported into mitochondria and processed into the mature subunit as efficiently as wild-type. Gel filtration analysis of the mitochondria revealed that at 10 min after import, markedly more K304E eluted as a monomer than did wild-type, and the amount of K304E tetramer formed was distinctly less than wild-type at any point up to 60 min after import, indicating that the assembly of K304E is defective. After further incubation, K304E decayed more rapidly than did wild-type, indicating a reduced stability. In similar studies, K304R behaved like the wild-type, while K304D closely resembled K304E, indicating that the presence of a basic residue at 304 is essential for tetramer formation and intramitochondrial stability of mature MCAD.     

Enzyme reamination of phosphororganic analogs of aspartate and glutamate

Phosphonous and phosphonic analogues of aspartate and glutamate are substrates of semireaction of enzymatic transamination catalysed by aspartate aminotransferase.     

Conversion of glutamate into aspartate in guinea-pig cerebral-cortex slices

1. When guinea-pig cerebral-cortex slices were incubated with [U-(14)C]glutamate as substrate, the specific radioactivities of the citric acid-cycle intermediates were lower than that of the aspartate isolated from the same vessels. 2. Aspartate was significantly labelled when [5-(14)C]glutamate was used as substrate and the aspartate contained almost no label when [1-(14)C]glutamate was present as substrate. 3. When specifically labelled glutamate was used as substrate, the label was found in the isolated aspartate in the position that would be predicted by citric acid-cycle mechanisms. 4. The results are consistent with the theory of ;compartmentation' of amino acid metabolism.     

Re-activation by glutamate or aspartate of amino-oxyacetate-inhibited aspartate aminotransferase in vitro and in isolated hepatocytes

Experiments with isolated rat hepatocytes and with cell extracts indicate, in contrast with previous reports, that pyruvate does not block or reverse the inhibition of aspartate aminotransferase (EC 2.6.1.1) by amino-oxyacetate. That inhibition, however, is partially overcome by glutamate or aspartate either in cell extracts or in whole cells incubated with substrate combinations that cause accumulation of those amino acids.     

A new subfamily of bacterial glutamate/aspartate receptors

The specificity of bacterial nutrient importers of the ATP-binding cassette (ABC) type depends on external receptor proteins that not only bind the solute to be transported, but also initiate the transport process by inducing ATP hydrolysis in the cytoplasmic nucleotide-binding domains. Here we propose a mode of ligand binding to the solute-binding protein AatJ that is required for glutamate uptake by the AatJMQP transporter in Pseudomonas putida KT2440. A homology model of the AatJ-glutamate complex was constructed using the E. coli glutamine-binding protein GlnH as the template. The general validity of the model was then confirmed by alanine scanning mutagenesis of several residues predicted to interact with the ligand and by semi-quantitative binding studies with [(14)C]-Glu and [(14)C]-Asp. A database search indicated that AatJ is a member of a distinct subfamily of the family 3 solute-binding proteins with specificity towards glutamate and aspartate.     

Distribution of gamma-aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat

The distributions of gamma-aminobutyric acid (GABA), glycine, glutamate and aspartate were measured in cochlear nuclei of two rats by quantitative histochemical mapping procedures. The levels and distributions in the two rats were comparable, and resembled those previously reported for cat cochlear nucleus. The results are consistent with a concept that these putative transmitter amino acids have similar levels and distributions in the cochlear nucleus among mammals.     

Extracellular levels of brain aspartate, glutamate and GABA during an inhibitory avoidance response in the rat

The extracellular levels of aspartate, glutamate and GABA were measured by microdialysis, coupled with an HPLC method, in rat prefrontal cortex (mPFC) and ventral hippocampus (VH) before and during the performance of a step-down inhibitory task. The basal levels of glutamate were about 50% higher than those of aspartate, and GABA levels were about 20-folds smaller than those of the excitatory amino acids. There were no significant differences in the basal levels of any of the three amino acids between the two brain regions. The extracellular levels of aspartate increased during acquisition and recall trials in both VH and mPFC, whereas those of glutamate increased in the VH during acquisition only. A significant increase in GABA levels was also detected during acquisition but only in the mPFC. The neuronal origin of the increased extracellular levels of aspartate, glutamate and GABA was demonstrated by administering tetrodotoxin directly into the mPFC or VH by reverse dialysis. These findings, together with previous evidence from our and other laboratories, indicate a differential release of aspartate and glutamate from excitatory neurons during the performance of behavioral responses, and therefore, distinct roles for the two excitatory amino acids should be envisaged.