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At least 937 records · Page 52Linked to original sources
PubMed1998

Postischemic enhancements of N-methyl-D-aspartic acid (NMDA) and non-NMDA receptor-mediated responses in hippocampal CA1 pyramidal neurons.

Glutamate receptor-mediated responses were investigated by using a whole-cell recording and an intracellular calcium ion ([Ca2+]i) imaging in gerbil postischemic hippocampal slices prepared at 1, 3, 6, 9, 12, and 24 hours after 5-minute ischemia. Bath application of N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate showed that NMDA-, AMPA- and kainate-induced currents were enhanced in postischemic CA1 pyramidal neurons at 1 to 12 hours after 5-minute ischemia. NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSC) were examined in postischemic CA1 pyramidal neurons at 3 hours after 5-minute ischemia to confirm whether synaptic responses are enhanced in the postischemic CA1 pyramidal neurons. The amplitudes of NMDA- and non-NMDA-receptor-mediated EPSC were enhanced in the postischemic CA1 pyramidal neurons. NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were also examined to determine whether the enhancement of currents is accompanied by the enhancement of [Ca2+]i elevation. The enhancements of NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were shown in the postischemic CA1. These results indicate that NMDA and non-NMDA receptor-mediated responses are persistently enhanced in the CA1 pyramidal neurons 1 to 12 hours after transient ischemia, and suggest that the enhancement of glutamate receptor-mediated responses may act as one of crucial factors in the pathologic mechanism responsible for leading postischemic CA1 pyramidal neurons to irreversible neuronal injury.

Animals↗
PubMed2001

Potential tuberculostatic agents: micelle-forming copolymer poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid.

With the objective of obtaining slow-acting isoniazid derivatives, of potential use as chemoprophylactics or chemotherapeutics in tuberculosis, the micelle-forming copolymer of poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid was synthesized. The derivative obtained was found to be active in Mycobacterium tuberculosis culture, with a minimal inhibitory concentration (MIC) 5.6 times lower than that of the tuberculostatic drug.

Antitubercular Agents↗
PubMed1997

N-methyl-DL-aspartic acid lesions of the medial preoptic area disrupt ongoing parental behavior in male rats.

The effects of axon-sparing, neurotoxic lesions of the medial preoptic area (MPOA) with N-methyl-DL-aspartic acid (NMA) on previously established parental behavior in male rats were investigated. Adult, sexually-inexperienced male rats were gonadectomized and seven days later implanted sc with a single estradiol (E2)-filled Silastic capsule on treatment Day 1. Three progesterone (P4) capsules were implanted sc on treatment Day 3 and removed on Day 21, one day prior to the start of behavioral testing. Males were tested daily with foster pups in order to induce parental behavior, i.e., contacting the test pups, pup retrieval, grouping, and crouching over three foster pups. Full parental behavior appeared in these males after an average of 3 days. After testing on the third consecutive day of parental behavior, parental males were infused bilaterally with either NMA or vehicle into the MPOA. NMA infusions resulted in a significant decline in all components of parental behavior by the next test session, a deficit which persisted throughout the 5 days of post-infusion testing. In contrast, parental care continued to be displayed in animals given vehicle infusions. These findings demonstrate that the cells in the MPOA play an important role in regulating ongoing parental care in male rats and indicate that the neural substrates controlling parental behavior in male and female rats are similar.

Animals↗
PubMed1998

Gonadotropin-releasing hormone (GnRH) gene regulation by N-methyl-D-aspartic acid in GT1-1 neuronal cells: differential involvement of c-fos and c-jun protooncogenes.

The present study examined the regulatory mechanisms of GnRH gene expression by N-methyl-d-aspartic acid (NMDA) in immortalized hypothalamic GnRH neurons (GT1-1 cells). NMDA (100 microM) stimulated GnRH mRNA levels transiently at 2 h after treatment. Dose-response experiment showed that there was a biphasic action of NMDA on GnRH mRNA levels: GnRH mRNA levels were increased by NMDA at lower concentrations (10 and 100 microM), but not at higher concentrations (1 and 10 mM). NMDA (100 microM)-induced GnRH mRNA levels were efficiently blocked by pre-treatment with NMDA receptor antagonists, MK-801 and AP-5. We next examined the signal transduction pathways involved in NMDA-induced GnRH gene expression based on previous findings that NMDA signal propagates into the cell through Ca2+ and nitric oxide (NO) pathways in many neurons. While ionomycin, a Ca2+ ionopore, application failed to alter GnRH gene expression, treatment of GT1-1 cells with sodium nitroprusside (SNP), an NO donor, increased GnRH gene expression with a similar time course to NMDA treatment. Moreover, application of GT1-1 cells with nitric oxide synthase (NOS) inhibitors (l-NAME, d-NAME, and NA) prior to NMDA treatment, inhibited NMDA-induced GnRH gene expression. These results indicate that the effect of NMDA is mediated by the NO signalling cascade. The mouse GnRH promoter activity was also increased by NMDA at low concentration (100 microM), but not at high concentration (1 microM), confirming the biphasic action of NMDA on GnRH mRNA levels. Since NMDA (100 microM) and SNP (1 microM) markedly induced c-jun expression, but not c-fos expression, we hypothesized that Jun activation is responsible for the transcriptional activation of GnRH gene expression. To examine this, we performed two different experiments. Treatment of NMDA greatly increased the activity of heterologous promoter of Fos/Jun responsive sequence (-187/-69) from the mouse GnRH promoter fused to hsv-tk minimal promoter. Moreover, overexpression of c-jun induced GnRH promoter activity, while c-fos overexpression decreased GnRH promoter activity. Taken together, this study indicates that NMDA regulates GnRH gene expression in GT1-1 cells through the NO-Jun signal transduction pathway.

Animals↗
PubMedDate not supplied

The effect of an arginine-glycine-aspartic acid peptide and hyaluronate synthetic matrix on epithelialization of meshed skin graft interstices.

Keratinocytes and fibroblasts interact with proteins of the extracellular matrix such as fibronectin and vitronectin through RGD (arginine-glycine-aspartic acid) cell-attachment sequences. This study evaluated the ability of a provisional synthetic matrix composed of an RGD peptide and hyaluronic acid to accelerate the epithelialization of the interstices of meshed, human, split-thickness skin when placed on full-thickness wounds of athymic mice. Full-thickness skin defects, sparing the panniculus carnosus, were created on athymic mice and 3:1 meshed, human skin was placed on them. The grafts had four central, isolated interstices, which epithelialized by migration of human keratinocytes. Conditions were either the addition to the wound of the synthetic matrix or a matrix of hyaluronic acid alone. The time to closure of the graft interstices was decreased (p < 0.02) in the wounds treated with the RGD peptide-hyaluronic acid provisional matrix. The resultant epithelium of the closed interstices was significantly thicker 8 days after surgery for the RGD-treated wounds. Basement membrane proteins (laminin and type IV collagen) were also found to be present at the dermoepidermal junction earlier in the RGD-treated wounds. These results imply that use of the RGD peptide conjugate to effect cell-matrix interactions may have clinical significance in the field of wound healing.

Animals↗
PubMed1992

Poly-L-aspartic acid does but triiodothyronine does not protect against gentamicin-induced cytotoxicity in the porcine kidney cell line LLC-PK1.

This study investigated the protective effect of thyroid hormone and poly-L-aspartic acid (PAA) in an in vitro model of gentamicin nephrotoxicity. LLC-PK1 porcine renal cells were grown in Medium 199 supplemented with either fetal bovine serum or thyroid hormone-depleted fetal bovine serum. After a preincubation with or without 30 nM L-triiodothyronine for 3 days, or 0.1 mM PAA for 1 hr, cells were coincubated with 1 mM gentamicin for an additional 3 days. Determinations were made of the following indicators of cell damage and/or viability: the numbers of detached dead cells, the total lactate dehydrogenase activity and its percentage release and gamma-glutamyl transpeptidase activity. Preincubation with L-triiodothyronine did not protect from gentamicin-induced cell death but did reduce cellular accumulation of gentamicin (3.2 +/- 0.8 micrograms/mg of protein vs. 5.2 +/- 1.8 micrograms/mg of protein in controls; P less than .05). In contrast, preincubation with 0.1 mM PAA decreased gentamicin-induced cell death (gentamicin: 685 +/- 416% of control dead cells and 487 +/- 48% of control lactate dehydrogenase release; PAA + gentamicin: 164 +/- 63% of control dead cells and 257 +/- 85% of control lactate dehydrogenase release; P less than .05) but failed to attenuate inhibition by gentamicin of gamma-glutamyl transpeptidase activity (gentamicin: 69 +/- 7% of control; PAA+gentamicin: 76 +/- 3% of control) and failed to alter cellular gentamicin levels. Protection against gentamicin nephrotoxicity by L-triiodothyronine was not demonstrated in LLC-PK1 cells, indicating that its protective effect in vivo is likely due to a systemic effect of the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Role of aspartic acid 121 in human pancreatic ribonuclease catalysis.

Bovine pancreatic ribonuclease (RNase A) is one of the most well studied enzymes of the ribonuclease family, unlike its human counterpart, the human pancreatic ribonuclease (HPR), whose physiological role in the body is not clearly understood. Human pancreatic ribonuclease consists of 128 amino acids and the main residues located in the active site of RNase A are also conserved in HPR. In the current study, to investigate the role of Asp-121 in the catalytic activity of human pancreatic ribonuclease, several variants were generated in which Asp-121 was either mutated to an alanine or C-terminal residues beyond Asp-121, and Phe-120 were deleted. The HPR mutants were cloned, expressed in E. coli and purified to homogeneity, and functionally characterized. The mutation D121A in HPR significantly decreased the rate of the enzymatic reaction, however this decrease was not universally observed for all substrates studied. Removal of the seven C-terminal amino acid residues thereby exposing Asp-121 yielded an HPR mutant with enhanced activity, however a further deletion removing Asp-121 resulted in the complete inactivation of HPR. Our results indicate that Asp-121 is crucial for the catalytic activity of HPR and may be involved in the depolymerization activity of the enzyme.

Alanine↗
PubMed1996

Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

Amino Acid Sequence↗
PubMed1988

Both competitive and non-competitive antagonists of N-methyl-D-aspartic acid disrupt brightness discrimination in rats.

Rats were trained to avoid or escape electric shocks in a symmetrical Y-maze by choosing to enter the brighter of two arms. Pretreatment with phencyclidine-like compounds disrupted brightness discrimination with greatly increased spontaneous locomotor activity between trials. The competitive antagonists of NMDA, 2-amino-7-phosphonoheptanoate (AP7) or 3-(+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) also disrupted brightness discrimination when injected into the cerebral ventricles, with no increase in movements between trials. The results suggest that the competitive antagonists of NMDA may impair sensory and cognitive functions in a manner similar to that produced by the phencyclidine-like compounds.

2-Amino-5-phosphonovalerate↗
PubMed2002

Anionic substitutes for catalytic aspartic acids in phosphoribulokinase.

Mutagenic substitution of the invariant D42 and D169 residues in phosphoribulokinase (PRK) with amino acids that contain neutral side chains (e.g., alanine or asparagine) results in large decreases in catalytic efficiency (10(5)- and 10(4)-fold for replacement of D42 and D169, respectively). To further evaluate the importance of anionic side chains at residues 42 and 169, substitutions of glutamic acid (D42E, D169E) and cysteine (D42C and D169C in an otherwise cysteine-free protein) have been engineered. All purified mutant enzymes bind the fluorescent alternative substrate trinitrophenyl-ATP and the allosteric effector NADH similarly to wild-type PRK. For D42E and D42C, V(max) exhibits substantial decreases of 135- and 220-fold, respectively. Comparable substitutions for D169 result in smaller effects; D169E and D169C exhibit decreases in V(max) of 39- and 26-fold, respectively. Thus, regardless of the type of substitution, changes at D42 more profoundly affect catalytic rate than do comparable changes at D169. Precedent with enzymes in which cysteine replaces an acidic residue suggests that oxidation of the thiolate to a sulfinate can convert low-activity cysteine mutants into enzymes with improved activity. Periodate oxidation of cysteine-free PRK results in a slight decrease in activity. In contrast, comparable treatment of D42C and D169C proteins increases activity by 5- and 7-fold, respectively. Thus, for reasonably efficient catalysis, PRK requires anionic character in the side chains of residues 42 and 169. The enzyme can, however, tolerate substantial structural and chemical variability at these residues.

Adenosine Triphosphate↗
PubMed2001

Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.

Allosteric Regulation↗
PubMed1983

Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

The following peptide was synthesized by classical methods in solution: Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-8). The Michaelis-Menten parameters for the hydrolysis of the Arg-Gly bond in F-8 by thrombin were determined to be Kcat = 31 X 10(-11) M [(NIH unit/L) s]-1 and KM = 310 X 10(-6) M. Comparison of these values with those determined previously for native fibrinogen and for a series of similar synthetic peptides, together with information about the amino acid sequences of this portion of the A alpha chain of abnormal fibrinogens, suggests an important role for Asp at position P10. Differences in the Michaelis-Menten parameters between F-8 and the 51-residue N-terminal CNBr fragment of the A alpha chain of fibrinogen correspond to only 1-2 kcal/mol in binding affinity.

Amino Acid Sequence↗
PubMed1985

Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101.

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236.

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.

Amino Acid Sequence↗
PubMed1987

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

Amino Acid Sequence↗