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(-)-Menthone, an inexpensive chiral auxiliary, was used to prepare both enantiomers of alpha-amino acids, amino alcohols, or oxazolidinones. The sequence includes the S(N)2' displacement by a cuprate reagent and a Curtius rearrangement as key steps.
The Xenopus laevis oocyte expression system was used to study the molecular composition of mRNAs encoding acidic amino acid (AA) receptors from rat brain. Xenopus oocytes injected with poly(A) mRNA express two general classes of AA receptors. One class consists of AA-gated cation channels. Responses are evoked by N-methyl-D-aspartate (NMDA), by kainate, and to a lesser extent by L-glutamate or quisqualate. The second class of receptor is coupled to an intracellular second messenger pathway activating an oocyte-encoded Ca2+-activated Cl- conductance. This second messenger-coupled AA receptor can be activated by L-glutamate or quisqualate. DL-2-amino-5-phosphonopentanoic acid and D-alpha-aminohexanedioic acid inhibit the AA-gated cation conductances activated by NMDA or kainate with different potencies but do not inhibit the second messenger-coupled AA receptor. Responses to NMDA are enhanced by micromolar level of glycine and are inhibited by Mg2+, Zn2+, or MK-801. Dose-response analysis reveals that the AA-gated cation conductance activated by kainate requires the binding of two agonist molecules. To study the molecular composition, the mRNAs were size-fractionated by denaturing agarose gel electrophoresis. About 20-fold purification in specific activity (nA/ng of mRNA injected) of mRNAs encoding the second messenger-coupled AA receptor was achieved. In contrast, only a slight enrichment of the mRNAs encoding the AA-gated channel was observed. This suggests that the second messenger-coupled AA receptor is encoded by a single size class of mRNA, whereas the AA-gated cation channel(s) is encoded by multiple species of mRNAs or by mRNAs whose size distribution is heterogeneous.
Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelled L- or D-aspartate, L-glutamate, L-pyroglutamate, or N-methyl-D-aspartate. L-gamma-Carboxyglutamate as well as the other amino acids were added in the unlabelled form. Results. L- and D-Aspartate (0.073 mmol X 1(-1)) are quickly resorbed at about the same rate. D-Aspartate resorption was blocked by L-aspartate (5 mmol X 1(-1)) but not by beta-alanine (5 mmol X 1(-1)). L-Aspartate resorption was inhibited by L-glutamate (2 mmol X 1(-1)) but not by D-glutamate, L-asparagine, L-phenylalanine or by succinate (2 mmol X 1(-1), each). The fast resorption of L-glutamate (0.073 mmol X 1(-1)) was blocked by D-aspartate, L-cysteate (2 mmol X 1(-1)), but not by 3-mercaptopicolinic acid (0.15 mmol X 1(-1)), L-glutamine, 2-oxoglutarate, taurine, N-methyl-L-glutamate or kainic acid (2 mmol X 1(-1), each). L-gamma-Carboxyglutamate (0.66 mmol X 1(-1)) and N-methyl-D-aspartate (2 mumol X 1(-1)) were found to be resorbed only at an extremely small rate. L-Pyroglutamate (0.076 mmol X 1(-1)) resorption was not influenced by L-glutamate (1 mmol X 1(-1)). Fractional excretion of gamma-carboxyglutamate was 7-25% (L-form) or 45-70% (D-form) at an artificially elevated plasma level of 12 mumol X 1(-1). It is concluded that L- and D-aspartate, L-glutamate, L-cysteate and, to a much smaller extent, L-gamma-carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for "acidic" amino acids. N-Substitution, the amidation of the beta- or gamma-carboxyl group, or the removal of the alpha-amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or gamma-carboxylated derivatives of "acidic" amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.
We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.
Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.
Amino acid sequence comparison of transposases belonging to the Tn3 family demonstrates that most of them cluster around three well known transposons, Tn21, Tn3, and Tn917. Eleven amino acid residues, including a triad of acidic residues, are invariant in the COOH-terminal third of the transposase protein while only five residues are invariant in the rest of the protein. We suggest that the invariant triad of Asp689, Asp765, Glu895 (numbering as in Tn3) may correspond to the D-D-35-E motif previously implicated in the catalytic performance of numerous transposases.
The properties of excitatory amino acid receptors in hippocampal slices were analyzed using agonist-induced stimulation of 22Na efflux rate. Several amino acids (L- and D-glutamate, N-methylaspartate) produce progressively smaller responses upon successive applications, whereas D,L-homocysteate does not. Several lines of evidence suggest that depletion of an intracellular pool of 22Na is not responsible for the apparent desensitization. Addition of the amino acids in the presence of an antagonist does not affect the response of the slices to subsequent applications, indicating that desensitization is dependent upon the interaction of the agonist with its receptor. The antagonist D-alpha-aminoadipate discriminates between various excitatory amino acids, completely blocking the responses to N-methylaspartate, D-glutamate, and D,L-homocysteate; partially antagonizing those of quisqualate and kainate; and being without effect on L-glutamate. The order of potency of several excitatory amino acids on the stimulation of 22Na efflux rate in hippocampal slices is highly correlated with their relative effects measured with electrophysiological techniques, but does not correlate with their relative potencies to inhibit [3H]glutamate binding to hippocampal membranes. The similarities in the properties of excitatory amino acid receptors evidenced with the 22Na efflux assay or with the electrophysiological approach in the in vitro hippocampal slice preparation indicate that the same receptors are sampled by the two techniques. The results are discussed in terms of a classification of these receptors into four different groups: a synaptic receptor, activated by D,L-homocysteate (tentatively defined as a G1 receptor), an extrasynaptic glutamate receptor (defined as a G2 receptor), an N-methylaspartate receptor, and a kainate receptor.
Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the hydrolysis of beta-(1, 4)-glycosidic bonds in chitin, the major structural polysaccharide present in the cuticle and gut peritrophic matrix of insects. Two conserved regions have been identified from amino acid sequence comparisons of family 18 glycosyl hydrolases, which includes Manduca sexta (tobacco hornworm) chitinase as a member. The second of these regions in M. sexta chitinase contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. In this study the functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins, D142E, D142N, D144E, D144N, E146D and E146Q, as well as the wild-type enzyme, were produced using a baculovirus-insect cell line expression system. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic and substrate binding properties were determined. Circular dichroism spectra of the mutant proteins were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was required for enzymatic activity because mutants E146Q and E146D were devoid of activity. D144E retained most of the enzymatic activity, but D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity relative to the wild-type enzyme. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicate that E146 probably functions as an acid/base catalyst in the hydrolytic mechanism, as do homologous residues in other glycosyl hydrolases. D144 apparently functions as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.
Proximate nutrients, gross energy content, mineral, amino acid and fatty acid composition were determined for teleost, cephalopod and crustacean prey of the Hawaiian monk seal. Crude protein was highest in the octopus, Octopus cyanea (80.0%), crude fat was highest in the Muraenid teleost, Gymnothorax eurostus (14.1%), whereas crude ash was highest in the lobster, Panulirus marginatus (11.6%). Gross energies ranged from 4.0 +/- 0.01 kcal g-1 in the Labrid teleost Bodianus bilulunatus to 6.0 +/- 0.12 kcal g-1 in the moray eel, Gymnothorax undulatus. Essential amino acids occurred in lower concentrations as a percentage of the total amino acids (35.8 +/- 2.6%) than non-essential amino acids (64.2 +/- 2.6%), but the ratio of individual amino acids to total amino acid concentrations were similar to those required by some monogastric terrestrial species and fingerling salmon. The fatty acid concentrations varied widely among species (range = 1.2-16.5 mg 100 mg-1); however, the teleosts had higher total fatty acids than the non-teleosts. This study indicates that, from a nutritional standpoint, some prey may be more beneficial to the Hawaiian monk seal; however, these prey are not necessarily the most abundant or available to some populations of the monk seal.
For elucidation of the structural and conformational requirements on the endotoxic and antagonistic activity of lipid A derivatives, we designed and synthesized lipid A analogues containing acidic amino acid residues in place of the non-reducing end phosphorylated glucosamine. Definite switching of the endotoxic or antagonistic activity was observed depending on the difference of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.
Recent estimates of molecular weight and cDNA sequencing indicate that smooth muscle caldesmons are considerably smaller than previously thought. The anomalous behaviour of these proteins during SDS-polyacrylamide gel electrophoresis can be correlated with their high acidic amino acid content. The results suggest a need to re-evaluate the stoichiometric relations of caldesmon to tropomyosin and actin in thin filaments and its presumed 1:1 interaction with calmodulin.
To identify the structural determinants necessary for mu opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr394 is required for agonist-induced early desensitization. To test whether Thr394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr383, since complete desensitization was achieved by restoring Thr383 (ATT). These results suggest that Thr394 is the primary recognition site for G protein-coupled receptor kinases, but Thr383 is also required for complete agonist-induced desensitization. The specificity of Thr394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.
In the spinal cord of cats anaesthetized with pentobarbitone, the excitation of interneurones and Renshaw cells by acidic amino acids or acetylcholine (Renshaw cells) is followed by a period of depressed excitability. This depression appears to be the consequence of prolonged repetitive firing rather than of the enzymic or chemical conversion of the excitants to neuronal depressants.
Feeding by the tobacco specialist Manduca sexta (Lepidoptera, Sphingidae) and application of larval oral secretions and regurgitant (R) to mechanical wounds are known to elicit: (a) a systemic release of mono- and sesquiterpenes, (b) a jasmonate burst, and (c) R-specific changes in transcript accumulation of putatively growth- and defense-related mRNAs in Nicotiana attenuata Torr. ex Wats. We identified several fatty acid-amino acid conjugates (FACs) in the R of M. sexta and the closely related species Manduca quinquemaculata which, when synthesized and applied to mechanical wounds at concentrations comparable with those found in R, elicited all three R-specific responses. Ion-exchange treatment of R, which removed all detectable FACs and free fatty acids (FAs), also removed all detectable activity. The biological activity of ion-exchanged R could be completely restored by the addition of synthetic FACs at R-equivalent concentrations, whereas the addition of FAs did not restore the biological activity of R. We conclude that the biological activity of R is not related to the supply of FAs to the octadecanoid cascade for endogenous jasmonate biosynthesis, but that FACs elicit the herbivore-specific responses by another mechanism and that the insect-produced modification of plant-derived FAs is necessary for the plant's recognition of this specialized herbivore.
Addition of D-aspartate, a substrate for the high-affinity transport of acidic amino acid transmitters, to suspensions of rat brain synaptosomes increased the rate of O2 consumption, uptake of 86Rb, and transport of 2-[3H]deoxyglucose. Stimulation of all three processes was abolished in the presence of ouabain. D-Aspartate had no effect on respiration in the medium in which NaCl was replaced by choline chloride. The ratio of the ouabain-sensitive increase in 86Rb uptake to that in O2 consumption was 12 to 1, which gives a calculated 86Rb(K+)/ATP of 2. It is concluded that electrogenic, high-affinity transport of sodium-D-aspartate into synaptosomes stimulates the activity of the Na+/K+ pump through an increase in [Na+]i.
A technique for the determination of organic acids, amino acids and sugars is described. The compounds of interest are separated by high-performance liquid chromatography (HPLC) and converted on-line by immobilized enzymes. The enzymes employed are covalently bound to a synthetic carrier. Hydrogen peroxide, which is produced in the reaction with oxidases, makes possible the application of an electrochemical detector. This arrangement combines the separation efficiency of HPLC, the substrate specificity of enzymes and the high sensitivity of electrochemical detection. The enzymes act according to known reaction mechanisms, but coupling with HPLC leads to a promising extension in the field of biosensors. The simple pretreatment of the samples (often a dilution step is sufficient) allows a rapid analysis of foodstuffs and biological or clinical extracts. The examples presented demonstrate the very high sensitivity of the method with detection limits in the nano- to picomolar range and a wide field of application.
Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide), a garlic-derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma-derived cell line (BJA-B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the GSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism.
The release of [3H]GABA from horizontal cells of goldfish retina was studied by biochemical analysis of perfused isolated retina. Retinas were incubated for 15 min in 0.72 microM [3H]GABA, rinsed for 30 min and then perfused with 1 min pulses of increasing concentrations of K+ and acidic amino acid agonists under a variety of conditions. Radioactivity in the perfusate was determined by liquid scintillation spectroscopy. The main findings are: (1) virtually all of the [3H]GABA released by L-glutamate (L-Glu) and L-aspartate (L-Asp) and 50% of the K+-evoked release, is calcium independent; (2) K+-evoked [3H]GABA release is only 10% of that released by L-Glu; (3) threshold [3H]GABA release occurs with 320 microM L-Glu, 1175 microM L-Asp, 4 microM quisqualic acid (QA), 4 microM kainic acid (KA) and 53 microM N-methyl-DL-aspartate (NMDLA); (4) the quisqualate antagonist glutamic acid diethyl ester (GDEE), has no specific inhibitory action on any of the agonists, whereas D-alpha-aminoadipic acid (D alpha AA), an NMDA antagonist, potently inhibits the action of NMDLA and L-Asp; (5) the presence of Mg2+, even at 1 mM, totally inhibits NMDLA and also inhibits the action of L-Glu and L-Asp below 1 mM; (6) D-Asp potentiates the action of L-Glu by 0.6-0.8 log units and completely inhibits the action of L-Asp; (7) L-Asp at a ratio of 3:1 potentiates the effect of L-Glu. From these and other results one concludes that: (a) [3H]GABA release from H1 cells is calcium independent and depends on factors other than passive depolarization, probably sodium; (2) the likely transmitter of red cones is L-Glu acting on quisqualate or kainate receptors, and (3) L-Asp acts predominantly on NMDA receptors and may provide a modulatory role in the outer retina by potentiating the action of L-Glu.