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PubMed1995

Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.

Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons. In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured. In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1. In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons. In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9. The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy [Li et al. (1993) Biochemistry 32, 5800-5808], indicates that the pKa > 9 in reduced thioredoxin is that of Asp26. This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 [Langsetmo et al. (1991) Biochemistry 30, 7603-7609]. The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1996

Structural and functional significance of aspartic acid 89 of the troponin C central helix in Ca2+ signaling.

The central helix of troponin C is highly conserved in length and amino acid sequence. In this region, D89 is conserved and specific to TnC. To investigate its significance, three mutations were made in avian fast troponin C: (1) D89 was replaced with A (D89A); (2) the central helix was replaced with a designed alpha-helix (alpha h89A) consisting of 87AEAALKAAMEA97; and (3) A89 of alpha h89A was replaced with D (alpha h89D). D89A and alpha h89A activated the regulated actomyosin ATPase poorly in the presence of Ca2+ (24 +/- 1.0% and 14 +/- 2.0%, respectively, of the wild type maximal activity) whereas alpha h89D had higher activity (113 +/- 3%). Both alpha h89A and D89A had apparently normal interactions with TnI and TnT whereas alpha h89D formed a complex with TnT even in the absence of Ca2+. The central helix was also replaced with a flexible random coil and rigid polyproline linkers in which D89 was Arg or Pro, respectively. Like alpha h89A and D89A, both mutants were defective in activation of the actomyosin ATPase in the presence of Ca2+. Changes in regulatory function of the mutants did not correlate with altered Ca2+ affinity, altered conformational changes upon binding divalent cations, or Ca(2+)-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca(2+)-dependent signal transduction, an event that can be dissociated from Ca(2+)-dependent binding to TnC targets on the thin filament.

Animals↗
PubMed1986

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.

Acetylglucosamine↗
PubMed2003

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits.

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.

Aspartic Acid↗
PubMed1987

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu.

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed2006

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue.

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.

Adenosine Triphosphatases↗
PubMed1997

Switch of coenzyme specificity of mouse lung carbonyl reductase by substitution of threonine 38 with aspartic acid.

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2'-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the kcat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2'-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

Alcohol Oxidoreductases↗
PubMed1988

Conserved aspartic acid residues 79 and 113 of the beta-adrenergic receptor have different roles in receptor function.

Deletion mutagenesis experiments have demonstrated that the binding site of the beta-adrenergic receptor involves the hydrophobic core of the protein (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). Single amino acid replacements for the conserved Asp79 and Asp113 within this putative transmembrane region had profound effects on the ability of the receptor to bind radiolabeled ligands (Strader, C. D., Sigal, I. S., Register, R. B., Candelore, M. R., Rands, E., and Dixon, R. A. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4384-4388). In this report we have analyzed the ability of these mutant receptors to stimulate adenylyl cyclase in the presence of agonists. The substitution of Asp79 with Ala caused 10-fold increases in both the Kd for isoproterenol binding and the Kact for adenylyl cyclase stimulation. The substitution of Asp113 by Asn or Glu resulted in 8,000-40,000 and 300-1,500-fold increases, respectively, in the Kact values for agonist stimulation of adenylyl cyclase without altering the maximum level of stimulation. Whereas the binding of antagonists to the receptor was not affected by substitution of Asp79, substitution of Asp113 decreased the affinity for the antagonist propranolol by 10,000-fold. These data are consistent with overlapping but not identical binding sites for agonists and antagonists on the beta-adrenergic receptor, in which the carboxylate group of Asp113 interacts with the amino group of the ligand. The sequence similarity among the family of G-protein-linked receptors suggests that the presence of an Asp residue at the analogous position of one of these receptors is predictive of the ability of the receptor to bind amines as ligands.

Animals↗
PubMed1981

Binding of porcine pancreatic phospholipase A2 to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process.

The interaction of porcine pancreatic phospholipase A2 (PA2) with micelles of various single-chain phospholipid analogues was studied by ultraviolet absorption difference spectroscopy and light-scattering measurements. The phospholipids used were either substrate analogues or products, varying in hydrocarbon chain lengths and polar head groups. The results indicate that the enzyme forms a stable complex over a wide range of enzyme and lipid concentrations. From the equivalent "molecular weight" and from the lipid to enzyme molar ratio (N) of the micelle--enzyme complex, it can be calculated that complexes containing saturated hydrocarbon chain lipids generally consist of two enzyme molecules and half of the number of lipid monomers present in free micelles. The interaction forces between the enzyme and lipid monomers bound in the complex are mainly hydrophobic. Stronger binding is found when the essential cofactor Ca2+ is bound to the enzyme. pH-titration studies on the binding of native PA2 to aggregated lipid structures showed that at least one group with a pKA value of 6.25 is involved in the interaction with lipid micelles. At acidic pH, micelle binding is stronger than at neutral or alkaline pH. Alkylation of the active site residue His48 resulted in a shift of the pKA value to 4.6, while addition of Ca2+ appears to stabilize the micelle-binding conformation of both native and modified enzymes over a broad pH range (pH 4--9.5). From these observations it is suggested that both the Ca2+ binding residue Asp49 [Fleer, E. A. M., Verheij, H. M., & de Haas, G. H. (1980) Eur. J. Biochem. 113, 283--288] and His48 control micelle binding of the native enzyme. For optimal binding in the absence of Ca2+, a long-distance hydrogen bond between these two residues is required; this can be established via a water molecule. It is assumed that it is a proton of this "H bond" which is titrated with a pKA value of 6.25. When the "H bond" is absent, as in the alkylated enzymes, Asp49 alone controls micelle binding with a pKA of 4.6. These results, together with the effect of Ca2+ on micelle binding, indicate that it is not the "hydrogen bridge" between His48 and Asp49 which is of main importance for an optimum binding conformation of the enzyme but the effective charge in the microenvironment of Asp49. It is proposed that a negative charge on this carboxylate causes a conformational change of the enzyme which leads to a protein conformation lacking an active micelle binding site. Binding of Ca2+ or reprotonation neutralizes this negative charge and restores the enzyme's ability to bind micelles.

Animals↗
PubMed2002

Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions.

Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.

Aspartic Acid↗
PubMed1991

A highly reactive beta-galactosidase (Escherichia coli) resulting from a substitution of an aspartic acid for Gly-794.

The beta-galactosidases of several mutagenized strains of Escherichia coli K12 which grew on lactobionate were found to be heat labile. Sequence analysis of the lacZ gene (ligated into Bluescript) of one of these strains (E. coli REH4) showed that the only change in the amino acid sequence was a substitution of an Asp for Gly-794. This change caused a dramatic increase of the activity when lactose was the substrate. The kcat of the purified enzyme from E. coli REH4 (G794D-beta-galactosidase) with lactose as the substrate was five to six times as large as the kcat of the normal enzyme with lactose. Purified G794D-beta-galactosidase was, however, less stable to heat and also to chymotrypsin (which cleaves next to Trp-585) than was normal beta-galactosidase. G794D-beta-Galactosidase bound substrates and substrate analog inhibitors less well than did normal beta-galactosidase while planar transition state analog inhibitors were more strongly bound. The ability to bind 2-amino-D-galactose (a positively charged transition state analog inhibitor) was either unaltered or was decreased somewhat. The data showed that the alteration in structure caused an increase in the value of k2 (the rate constant for the step in which the glycosidic bond is cleaved) with each substrate tested (the increase was at least 25-fold when lactose was the substrate) while k3 was decreased about 4-fold (k3 is the rate constant for the common hydrolysis step with each substrate). Since k2 is rate determining when lactose is the substrate of the normal enzyme, the increase in k2 resulted in a large increase in rate despite the fact that the value of k3 decreased. Large rate increases were not found with the other two substrates because the k2 values were not increased by large factors and because the decrease in the value of k3 negated the effects of the increased k2 values. The destabilization of the substrate binding coupled with a stabilization of the binding of a planar transition state is a possible cause of the significant increase in the value of k2 and of the enhanced activity with lactose.

Aspartic Acid↗
PubMed1985

N-(Phosphonoacetyl)amino phosphonates. Phosphonate analogues of N-(phosphonoacetyl)-L-aspartic acid (PALA).

Michaelis-Arbuzov reaction of N-(chloroacetyl)amino phosphonic acids or their esters, followed by acidolysis, gives moderate yields of N-(phosphonoacetyl) derivatives of a variety of (aminoalkyl)phosphonic acids, including analogues of the cytostatic agent PALA, in which the alpha- or beta-carboxylic groups in the aspartate moiety are replaced by a PO3H2 function. Assay of cytostatic activity with human KB cell lines indicates that the substitution of any of the COOH groups in PALA with PO3H2 results in total loss of cytostatic activity. No activity was observed also in the case of other [N-(phosphonoacetyl)amino]alkylphosphonic acids described in this report.

Aspartic Acid↗
PubMed1967

Amino acid metabolism and protein synthesis in a pyrithiamine-requiring Staphylococcus aureus mutant.

1. Amino acid metabolism and protein synthesis in a Staphylococcus aureus mutant strain that requires pyrithiamine for optimum growth were studied and compared with those in the thiamine-requiring parent S. aureus. 2. The mutant strain utilized amino acids at a higher rate than did the parent strain. The utilization of glutamic acid, serine and glycine was much stimulated in the mutant strain. 3. The rate of oxidation of glutamic acid, aspartic acid, isoleucine and glycine was higher in the mutant strain. 4. The mutant strain contained serine, glycine, tyrosine, glutamic acid, aspartic acid, arginine and histidine as free amino acids, whereas the parent strain possessed lysine, arginine, histidine, aspartic acid and glutamic acid. 5. The mutant strain possessed slightly higher glutamate-oxalo-acetate transaminase activity, whereas the activities of glutamate-pyruvate transaminase were similar in both strains. 6. The incorporation of (14)C from [2-(14)C]-acetate into individual amino acids of the cell protein was greater in the mutant strain. 7. The incorporation of (14)C-labelled amino acids into the cell proteins of the mutant strain was not much different from that in the parent strain. 8. Induction of beta-d-galactosidase in the mutant strain did not occur, whereas induction of this enzyme is possible in the parent strain. Thiamine or pyrithiamine has no direct effect on the induction of beta-d-galactosidase.

Acetates↗
PubMed1984

Decrease in N-acetyl-L-aspartic acid in brain of myodystrophic mice.

Brain tissue from myodystrophic mice (male 129/ReJ-dy and female 129 B6F1/J-dy) was examined to determine whether CNS abnormalities accompany the known muscular defects. Brain N-acetyl-L-aspartate, aspartate, and glutamate were significantly lower than in brain from control mice. These amino acids are only slightly reduced in brains of female dystrophic mice.

Animals↗
PubMed2000

Developmental maturation of the N-methyl-D-aspartic acid receptor channel complex in postnatal rat brain.

The N-methyl-D-aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [(3)H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors - an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1-day 14) is highly sensitive to spermidine, L-glutamate alone potentiates [(3)H]MK-801 binding, and glycine failed to potentiate an L-glutamate-induced increase in [(3)H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [(3)H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [(3)H]MK-801 binding was not seen in the presence of L-glutamate alone, and L-glutamate and glycine or L-glutamate and spermidine or L-glutamate, glycine and spermidine together, significantly increased [(3)H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.

Age Factors↗
PubMed1987

Evidence for N-methyl-D-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog.

We have investigated the role of N-methyl-D-aspartate (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg2+ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-applied D-2-amino-5-phosphonovaleric acid (D-APV, 25-50 microM), a selective NMDA antagonist. In a low Mg2+ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as in their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and has a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of hte latter was reduced by D-APV (25-50 microM) to about 65% of the control value in the presence of 1 mM Mg2+. During bath application of NMDA (100 microM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg2+ concentration. Bath application of NMDA (0.1-1 microM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were simulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these simulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.

2-Amino-5-phosphonovalerate↗
PubMed2005

Endocrine roles of D-aspartic acid in the testis of lizard Podarcis s. sicula.

In the lizard Podarcis s. sicula, a substantial amount of D-aspartate (D-Asp) is endogenous to the testis and shows cyclic changes of activity connected with sex hormone profiles during the annual reproductive phases. Testicular D-Asp content shows a direct correlation with testosterone titres and a reverse correlation with 17beta-estradiol titres. In vivo experiments, consisting of i.p. injections of 2.0 micromol/g body weight of D-Asp or other amino acids, in lizards collected during the three main phases of the reproductive cycle (pre-reproductive, reproductive and post-reproductive period), revealed that the testis can specifically take up and accumulate D-Asp alone. Moreover, this amino acid influences the synthesis of testosterone and 17beta-estradiol in all phases of the cycle. This phenomenon is particularly evident during the pre- and post-reproductive period, when endogenous testosterone levels observed in both testis and plasma were the lowest and 17beta-estradiol concentrations were the highest. D-Asp rapidly induces a fall in 17beta-estradiol and a rise in testosterone at 3 h post-injection in the testis and at 6 h post-injection in the blood. In vitro experiments show that testicular tissue converted L-Asp into D-Asp through an aspartate racemase. D-Asp synthesis was measured in all phases of the cycle, but was significantly higher during the reproductive period with a peak at pH 6.0. The exogenous D-Asp also induces a significant increase in the mitotic activity of the testis at 3 h (P < 0.05) and at 6 h (P < 0.01). Induction of spermatogenesis by D-Asp is recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for proliferation cell nuclear antigen (PCNA). The effects of D-Asp on the testis appear to be specific since they were not seen in lizards injected with other D- or L-forms of amino acids with known excitatory effects on neurosecretion. Our results suggest a regulatory role for D-Asp in the steroido-genesis and spermatogenesis of the testis of the lizard Podarcis s. sicula.

Amino Acid Isomerases↗
PubMed1996

Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.

Amino Acid Sequence↗