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The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.

Phenylalanyl-tRNA synthetases from mitochondria of yeast and hen liver resemble their corresponding cytoplasmic counterparts. Whereas slight intraspecies differences at the amino acid binding site, reflecting variations in the structures of these distinct enzymes, are exploitable by phenylalanine analogues, no intraspecies difference can be noted for the strategies to achieve the high fidelity of protein synthesis. While the yeast mitochondrial enzyme follows the pathway of posttransfer proofreading, the hen liver mitochondrial enzyme uses a tRNA-dependent pretransfer proofreading in the case of the natural amino acids. The accuracy of mitochondrial phenylalanyl-tRNA synthetases appears to be even better than the accuracy of the corresponding cytoplasmic enzymes. Interspecies rather than intraspecies differences for the functional role of certain amino acid residues of the enzymes further indicate the close relationship of the intracellular heterotopic isoenzymes. By use of a highly sensitive immunospotting procedure, common antigenic determinants are detected only within the enzymes from the two intracellular compartments of the same organism. The results suggest the origin of the cytoplasm-mitochondrion isoenzyme pair by independent gene duplication of the ancestral nuclear gene. A similarity of mitochondrial enzymes to the phenylalanyl-tRNA synthetase from Escherichia coli is not observed.

A comparative study of the binding of L-cysteine sulfinic acid (CSA) and L-glutamic acid (GLU) to various subcellular fractions of membranes from rat brain was made. Kinetic parameters were determined in all fractions for both types of binding. The effects of membrane preincubation, freezing, and thawing were also examined. The GLU and CSA specific binding levels increased in medium-density (C) and high-density (D) synaptic membranes as compared to the crude mitochondrial/synaptosomal membranes (wP2). Freezing and thawing reduced CSA binding in all tested subcellular fractions. GLU binding is reduced in wP2, C, and D. Binding to the "light" synaptic membranes (B) was not significantly affected, suggesting the presence of two GLU sites. Kinetics of the GLU binding indicated that the temperature-sensitive and -insensitive sites have Kd of 600 nM and 1,100/nM, respectively. Preincubation of fresh membranes conversely affected CSA and GLU binding to the various subcellular fractions, increasing CSA binding in wP2, B, C and decreasing it in D suggesting the existence of distinct sites for GLU and CSA. Preincubation of previously frozen membranes similarly modified CSA and GLU binding except in B fractions. CSA and GLU binding exhibited different pH sensitivities in both fresh and frozen membranes. These results indicate that multiple acid amino acid binding sites exist in membranes and that they can be differentiated according to their sensitivity to temperature. They also suggest the existence of distinct sites for CSA and GLU in fresh membranes, giving further support to the hypothesis that CSA may also serve a neurotransmitter role in the rat central nervous system.

p -Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p -chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.

There is a great deal of information on the active transport of amino acids by Escherichia coli in the literature. Piperno and Oxender were the first to discover an amino acid binding protein. They noted that when E. coli is exposed to osmotic shock the transport of leucine and isoleucine is reduced; however, the uptake of alamine and proline remained unaffected. While the genetics and biochemistry of amino acid transport in E. coli are well studied, there is virtually no information in the literature on the transport of amino acids in Agrobacterium tumefaciens. In the present study, the active transport of proline and valine in Agrobacterium tumefaciens C-58 was studied. In addition, use of the same or different energy store in the two transport systems was studied. Apparently, there are two different mechanisms for the uptake of proline and valine in Agrobacterium tumefaciens. Proline uptake is enhanced by the carbon and energy source glucose, whereas the uptake of valine is inhibited by glucose. Proline uptake could be driven by either oxidative or substrate level processes. The substrate level pathway which was active with glucose was found to be insensitive to a membrane ATPase inhibitor, dicyclohexyl carbo-diimide, (DCC). Valine uptake, on the other hand, appeared to be sensitive to DCCD, a membrane ATPase inhibitor.

Determination of the molecular weight of valyl and isoleucyl tRNA synthetases under denaturing conditions leads to the conclusion that both enzymes are made of a single polypeptide chain of a molecular weight of 110000 approximately. The presence of one amino acid binding site per chain was determined by equilibrium dialysis.

Membranes obtained from exponentially growing yeast were extracted with buffered solution of 1% Tween 80 plus 0.1m MgCl2. The soluble proteins, saparated from detergent and fine membrane particles on Saphadex G-100 colunn, were fond, to have binding affinity for Val, Lau, Ileu, Asp, Lys, His, Arg, Phe and Met. The occurrence of individual binding activities in different preparations was irregular. This and the fast inactivation by aging showed the extrame sensitivity of the molecules of binding proteins towards deleterious agents. Saturation of the binding proteins with aspartic acid and phenylalanine was of an identical biphasic shape indicating the change ofKdiss and suggesting the existence of several binding sites for each amino acid. In our opinion the above listed observations imply low specificity of amino acid binding proteins in yeast.

The transfer RNA molecule is able to recognize both specific amino acids and corresponding genetic code words. In so doing, it translates the information encoded in nucleic acid messages into protein products. Multiple discrete steps in this process include amino acid binding, transport to the ribosome, binding to one ribosomal site, peptide bond formation, translocation to another ribosomal site, and release from the ribosome. Initiation of protein synthesis requires different tRNAs as compared to those utilized for the growth of initiated protein chains. Other reactions known to involve tRNA as a reactant include cell wall synthesis, phospholipid synthesis, phosphoserine synthesis, and repression of synthesis of certain amino acid metabolizing enzymes. Knowledge of the three-dimensional structure of tRNA molecules will provide a framework for integrating our understanding of these many metabolic functions.

nap , previously characterized by Jacobson and Metzenberg as a neutral and acidic amino acid transport mutant, was found in this study to be reduced in transport activity for a variety of metabolites. Analysis of glycoprotein molecules involved in amino acid binding indicates that the deficiency is not at this level. The deficiency appears to be in some common component of all active transport systems.

The kinetics of phenylalanyl-tRNA and seryl-tRNA formation were investigated with tRNAs and aminoacyl-tRNA synthetases from yeast. Phenylalanyl-tRNA synthetase yielded linear Lineweaver-Burk plots with tRNAphe, phenylalanine, and 1,N6-ethenoadenosine triphosphate (ɛATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5′-phosphate, phenylalanyl-tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver-Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites. Seryl-tRNA synthetase yielded a linear Lineweaver-Burk plot only with ɛATP as variable substrate. The Lineweaver-Burk plots for serine and tRNASer were non-linear; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses at least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules. The aminoalkyl adenylates, phenylalaninyl adenosine 5′-phosphate and serinyl adenosine 5′-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid. ɛATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather different kinetic properties. Modified tRNAphe and tRNASer, in which the 3′-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of ɛATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodified tRNAs, as fluorescent probes in synthetase-tRNA interaction studies. The kinetic behavior of phenylalanyl-tRNA synthetase appears to be much simpler than that of seryl-tRNA synthetase, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.

After removal of the terminal alanine from insulin by carboxypeptidase, it was impossible to demonstrate the recombination of alanine with the protein, using alanine-1-C14 as an indicator. The presence of the active enzyme in 20 % sucrose, either at normal atmospheric pressure or at a high pressure (6000 atm.) did not affect the uptake of alanine-1-C14 by the insulin. Experiments of this type yielded negative results in spite of varying the pH, time of reaction, and concentration of alanine over wide ranges. However, alanine, particularly at high pH, did become, firmly bound to insulin (and albumin) by a reaction which is mediated by heavy-metal ions. The dependence of this amino acid binding to protein on metal-ion concentration, pH, and time of reaction has been investigated. In addition to the previously mentioned results with respect to the recombination of alanine with insulin from which the terminal alanine had been removed, it has also been found impossible to demonstrate any exchange of alanine in insulin for alanine-1-C14 in solution in the presence of either carboxypeptidase or trypsin.

Quantitative receptor autoradiography was used to measure muscarinic cholinergic, benzodiazepine, kainate, phencyclidine (PCP), N-methyl-D-aspartate (NMDA) (measured in Tris acetate), quisqualate-sensitive, non-quisqualate-sensitive and total glutamate (measured in Tris chloride buffer) binding sites in adjacent sections of the hippocampal region of 10 Alzheimer's disease, nine control, and six demented, non-Alzheimer's disease postmortem human brains. The measurements were compared to the number of neurofibrillary tangles as revealed by Congo red staining of adjacent sections. All assays and measurements were done by observers blinded to the clinical diagnoses. Binding was decreased significantly for all ligands except quisqualate in stratum pyramidale of CA1 of the Alzheimer's disease brains. The binding loss was significantly greater for the non-quisqualate and NMDA sites than for the muscarinic, benzodiazepine and kainate sites with the total glutamate and PCP site losses being intermediate. Only the loss of benzodiazepine binding was significantly correlated with the number of neurofibrillary tangles. Lesser binding losses were seen in adjacent areas. This difference in the degree of binding decrease is consistent with the hypothesis that NMDA receptors are located on more distal dendrites of hippocampal neurons. There they may be relatively more vulnerable than the other receptors to the pathological process.

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms based on geometric factors and surface electrostatic potentials. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using both a cell population assay and single cell imaging. We then identify a potential amino acid binding site using computational methods and site-directed mutagenesis. The effect of amino acid binding in altering intracellular calcium responses, especially calcium oscillations, and its synergistic interaction with the effects of extracellular calcium on these parameters are also investigated. A common activation mechanism for CaSR and other family C GPCRs, such as mGluR1 by extracellular calcium and amino acid is been proposed. These results could have important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--one an inorganic divalent cation, the other a nutrient--and how the receptor senses these agonists in health and in disease states.