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PubMed2006

Nanocomposites of hydroxyapatite with aspartic acid and glutamic acid and their interaction with osteoblast-like cells.

The direct synthesis of hydroxyapatite (HA)-aspartic acid (ASP) and HA-glutamic acid (GLU) nanocrystals was carried out in presence of different amounts of the amino acids in solution. ASP and GLU incorporation into HA crystals reduces the coherent length of the perfect crystalline domains along the long dimension (002) and, even more, along the cross section (310) of the apatite crystals, suggesting a specific interaction of the amino acids with the HA structure. FTIR analysis indicates that the carboxylic groups of the acidic amino acids interact with the calcium ions of HA. The relative amount of ASP incorporation into HA nanocrystals is greater than that of GLU, suggesting a greater affinity of ASP for HA. Osteoblast-like, MG63, cells cultured on the composite nanocrystals display good proliferation and increased values of ALP activity, collagen type I, TGF-betaI and osteocalcin production, indicating that the presence of the acidic amino acids enhances osteoblast activation and extra-cellular matrix mineralization processes.

Aspartic Acid↗
PubMedDate not supplied

Accelerated healing and reduced need for grafting in pediatric patients with burns treated with arginine-glycine-aspartic acid peptide matrix. RGD Study Group.

Arginine-glycine-aspartic acid (RGD) peptide matrix is designed to promote dermal healing by providing a molecular scaffold that facilitates cell ingrowth and establishment of normal tissue architecture. This study investigated the effectiveness and safety of RGD peptide matrix in the treatment of partial-thickness scald burns in pediatric patients. Either topical RGD peptide matrix or control treatment with silver sulfadiazine was applied to matched burn sites daily for up to 21 days. With RGD peptide matrix application under synthetic occlusive dressing, the incidence of healing was nearly threefold higher, average time to healing 2.5 days shorter (regression estimation), extent burn closure at all treated sites 37% greater, and number of needed grafting procedures fourfold lower compared with control treatment. All these differences were statistically significant. RGD peptide matrix was well tolerated. RGD peptide matrix promoted and accelerated healing in this study and thus may be able to reduce morbidity and treatment costs of partial-thickness burns in pediatric patients.

Administration, Topical↗
PubMed1999

Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp. PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme.

The branched polypeptide multi-L-arginyl-poly-L-aspartic acid, also called cyanophycin, is a water-insoluble reserve material of cyanobacteria. The polymer is degraded by a specific hydrolytic enzyme called cyanophycinase. By heterologous expression in Escherichia coli, a gene encoding cyanophycinase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The gene, designated cphB, codes for a protein of 29.4 kDa. The high level of expression of active cyanophycinase in E. coli from the Synechocystis gene allowed for its purification to electrophoretic homogeneity. The enzyme, which appears to be specific for cyanophycin, hydrolysed the polymer to a dipeptide consisting of aspartic acid and arginine. Based on inhibitor sensitivity and primary sequence, cyanophycinase appears to be a serine-type exopeptidase related to dipeptidase E [Conlin, C.A., Haakensson, K., Liljas, A. & Miller, C.G. (1994) J. Bacteriol. 176, 166-172].

Amino Acid Sequence↗
PubMed2006

D-Aspartic acid and nitric oxide as regulators of androgen production in boar testis.

D-Aspartic acid (D-Asp) and nitric oxide (NO) are two biologically active molecules playing important functions as neurotransmitters and neuromodulators of nerve impulse and as regulators of hormone production by endocrine organs. We studied the occurrence of D-Asp and NO as well as their effects on testosterone synthesis in the testis of boar. This model was chosen for our investigations because it contains more Leydig cells than other mammals. Indirect immunofluorescence applied to cryostat sections was used to evaluate the co-localization of D-Asp and of the enzyme nitric oxide synthase (NOS) in the same Leydig cells. D-Asp and NOS often co-existed in the same Leydig cells and were found, separately, in many other testicular cytotypes. D-Asp level was dosed by an enzymatic method performed on boar testis extracts and was 40+/-3.6 nmol/g of fresh tissue. NO measurement was carried out using a biochemical method by NOS activity determination and expressed as quantity of nitrites produced: it was 155.25+/-21.9 nmol/mg of tissue. The effects of the two molecules on steroid hormone production were evaluated by incubating testis homogenates, respectively with or without D-Asp and/or the NO-donor L-arginine (L-Arg). After incubation, the testosterone presence was measured by immunoenzymatic assay (EIA). These in vitro experiments showed that the addition of D-Asp to incubated testicular homogenates significantly increased testosterone concentration, whereas the addition of L-Arg decreased the hormone production. Moreover, the inclusion of L-Arg to an incubation medium of testicular homogenates with added D-Asp, completely inhibited the stimulating effects of this enantiomer. Our results suggest an autocrine action of both D-Asp and NO on the steroidogenetic activity of the Leydig cell.

Animals↗
PubMed1983

Cation-exchange high-performance liquid chromatography of proteins on poly(aspartic acid)-silica.

A simple cation-exchange material for high-performance liquid chromatography of proteins was developed. Poly(succinimide) reacted rapidly with aminopropyl-silica and the product was hydrolyzed to poly(aspartic acid)-silica. Reaction conditions were optimized to yield a material with an ion-exchange capacity of 430 mg hemoglobin/g material. High-performance liquid chromatographic columns of the material featured excellent performance in terms of capacity, selectivity, recovery of enzyme activity, peak shape and durability. Protein standards and clinical hemoglobin samples were well resolved in minutes. Poly(succinimide)-silica was readily derivatized to give products other than poly(aspartic acid)-silica, and several such materials were prepared. Such materials could be useful for affinity chromatography or enzyme immobilization.

Chromatography, High Pressure Liquid↗
PubMed2002

A new pathway to aspartic acid from urea and maleic acid affected by ultraviolet light.

The photochemistry of a mixture of urea and maleic acid, which are thought to have been widely present on the primitive Earth, was studied in order to examine a possibility of the formation of amino acids. When an aqueous solution of urea and maleic acid was irradiated with an ultraviolet light of wavelength 172 nm, urea was revealed to be rather resistant to photochemical decomposition. In contrast, maleic acid was completely decomposed within 4 h, reflecting the reactivity of a C-C double bond in the molecule. In the reaction mixture, 2-isoureidosuccinic acid was detected. The acid was considered to be formed by addition of an isoureido radical which had been produced from urea by the action of a hydroxyl radical, to a C-C double bond of maleic acid. The isoureido group of the product was revealed to undergo thermal rearrangement to afford 2-ureidosuccinic acid (N-carbamoylaspartic acid). The result suggested a novel pathway leading to the formation of aspartic acid from non-amino acid precursors, possibly effected by UV-light on the primitive Earth. The formation of ureidocarboxylic acids is of another significance, since they are capable of undergoing thermal polymerization, resulting in formation of polyamino acids.

Aspartic Acid↗
PubMed1976

Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in Cyanobacteria.

The polypeptide contained in the cyanophycin granule, a characteristic cyanobacterial subcellular inclusion, is shown to be a highly branched structure consisting of a polyaspartic acid core to which arginyl residues are attached at each free carboxyl group of the polyaspartic acid. The evidence supporting such a model includes: (i) The resistance of the polypeptide to a variety of enzymatic procedures commonly used to degrade linear polypeptide chains. (ii) The inability to degrade the polypeptide from the amino terminal using sequential Edman degradation. (iii) The preferential relase of arginine following hydrolysis of the polypeptide in dilute acid (0.03 M acetic acid, 105 degrees C). (iv) The demonstration by chemical linkage analysis that both the carboxyl groups of aspartic acid are unavailable for reduction and must therefore by involved in covalent linkages and that many arginyl residues can be reduced and therefore must not be involved in covalent linkage. (v) The removal of approximately 75% of the arginine from the polypeptide by chemical treatment of the polypeptide using methods designed to cleave carboxyl-terminal amino acids. The highly branched structure of the cyanophycin granule polypeptide is similar in form to synthetically produced multichain polyamino acids, and using the nomenclature for describing multichain polyamino acids, it is proposed that the cyanophycin granule polypeptide be called multi-L-arginyl- -polyaspartic acid.

Amino Acid Sequence↗
PubMed1991

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding.

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.

Amino Acid Sequence↗
PubMed2006

High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody.

A method for rapid determination of free D-aspartic acid (D-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against D-Asp for the first time. An anti-D-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated D-Asp to BALB/c mice. The obtained antibody has a high specificity toward D-Asp but shows a slight cross-reactivity to all other D- and L- amino acids including L-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 micromol/mL D-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of D-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of D-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of D-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this D-amino acid.

Amino Acid Sequence↗
PubMed1999

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.

Apoenzymes↗
PubMed2005

Aspartic acid scanning mutation analysis of a goldfish growth hormone-releasing hormone (GHRH) receptor specific to the GHRHsalmon-like peptide.

Growth hormone-releasing hormone (GHRH) plays a pivotal role in the regulation of growth. The study of goldfish GHRH and its receptor is of particular interest as it is so far the only animal model in which two forms of GHRH-like (catfish-like and salmon-like) peptides coexist, and these peptides share only 30-40% of amino acid sequence identities with their mammalian counterparts. For these reasons, we have previously characterized a goldfish GHRH receptor, which is specific for a synthetic carp GHRH-like peptide. In this study, we investigated the structure-function relationships between the receptor and various ligands. Interestingly, among the two endogenous goldfish GHRH-like peptides, only the GHRHsalmon-like peptide was able to stimulate CHO cells transfected with the goldfish GHRH receptor. When the receptor was challenged by GHRHsalmon-like peptide either continuously for 45 min or periodically at 45-min intervals, mild homologous desensitization was observed. To determine whether the negatively charged residues of the receptor are responsible for discriminating GHRHsalmon-like from GHRHcatfish-like, 10 aspartic acid residues residing in the N-terminal ectodomain and the second exoloop were individually mutated to alanine by site-directed mutagenesis. Among these 10 mutants, four of them (D66A, D122A, D190A, and D196A) were defective as indicated by both cAMP assays and extracellular acidification rate measurements. Confocal microscopic studies showed that the D66A and D122A mutants, but not the D190A and D196A mutants, were expressed properly at the plasma membrane. Collectively, these results suggest that aspartic acid residues at positions 66 and 122 are critical for the interaction between the goldfish GHRH receptor and its endogenous ligands.

Animals↗
PubMed1983

Phase I and clinical pharmacological evaluation of biochemical modulation of 5-fluorouracil with N-(phosphonacetyl)-L-aspartic acid.

5-Fluorouracil (FUra) is a clinically useful antineoplastic agent. Preclinical studies suggest that the therapeutic effects of FUra can be enhanced by pretreatment with N-(phosphonacetyl)-L-aspartic acid (PALA), an inhibitor of aspartate transcarbamylase. The objective of treatment with PALA is to increase the activation of FUra by inhibiting the normal pathway of de novo pyrimidine biosynthesis. Theoretically, the optimal dose of PALA should produce effective blockade of this pathway without increasing toxic effects of FUra. Using pyrazofurin-induced orotic aciduria and orotidinuria as a measure of this pathway, it as determined that PALA (250 mg/sq m) is effective in inhibiting total-body pyrimidine synthesis. Sixty-eight adult patients with cancer were treated with combinations of PALA and FUra. High doses of PALA (1 to 2 g/sq m) prevented the use of full dosage of FUra; however, PALA (250 mg/sq m) can be administered 24 hr before FUra (750 mg/sq m) once weekly for at least 3 weeks. The toxicity observed using that combination of doses was mild to moderate myelosuppression, mucositis, diarrhea, nausea, and vomiting. Further clinical studies are warranted.

Adult↗
PubMed1997

Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.

Animals↗
PubMed1989

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase.

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.

Amino Acid Sequence↗
PubMed1999

[Protective effect of poly DL-aspartic acid on ototoxicity of gentamicin].

OBJECTIVE: To observe the protective effect of poly DL-aspartic acid (PAA) on ototoxicity of gentamicin (GM). METHODS: Fifty F-344 rats were divided into four groups, GM only PAA + GM, PAA only and saline control. ABR thresholds at different frequencies were measured at different times and hair cell losses were numerated. Two-dimensional diffusion assays in the culture medium were performed to evaluate the effect of PAA on the antimicrobial activity. RESULTS: Eighteen days after the treatment, ABR thresholds at 10 kHz and 8 kHz as well as hair cell losses in the GM-treated group showed significant differences as compared with other three groups (P < 0.01). CONCLUSION: PAA had the protective effect against the cochlear ototoxicity of GM without decreasing its antimicrobial activity.

Animals↗