Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: Y22D7AL .14
 53,079 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self-association of ATP via the concentration dependence of the 1H-NMR chemical shifts for H2, H8 and H1' in D2O at pD 8.4 (25 degrees C) in the range 0.0025-0.4 M in the presence and absence of poly(alpha, L-lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H.Lys)n+, whereas at pD 12.1 only approximately 10% of the epsilon-amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non-cooperative stacking. The stacking tendency follows the series: ATP4- (K = 1.3 M-1; pD 8.4) < ATP4-/poly(H.Lys)n+ (K = 11.5 M-1; pD 8.4) > ATP4-/90% poly(Lys)/10% poly(H.Lys)n+ (K = 3.1 M-1; pD 12.1). It is evident that poly(H.Lys)n+ assists the association of ATP by a factor of approximately 10, and it is suggested that, via its positively charged epsilon-ammonium groups, poly(H.Lys)n+ acts as a matrix by aligning ATP4- ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such as epinephrine or 5-hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides.
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PMID:The assisted self-association of ATP4- by a poly(amino acid) [poly(Lys)] and its significance for cell organelles that contain high concentrations of nucleotides. 885 48

Searching for novel approaches for uncoupling glycation from hyperglycemia as a cause of diabetic complications, a Pseudomonas sp. soil strain containing a membrane-bound enzyme that deglycates amino acids under release of free fructosamine was isolated (Gerhardinger, C., Marion, S. M., Rovner, A., Glomb, M., and Monnier, V. M. (1995) J. Biol. Chem. 270, 218-224). This enzymatic activity was found to be very sensitive to inactivation by most detergents. From the plasma membrane ( approximately 3 mg/ml protein concentration), the enzyme could be solubilized in active form using 10 mM 3-[(3-chlolamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate aided by 2 M NaCl and 10% glycerol (27% optimal solubilization yield). The supernatant from a 55% saturation (NH4)2SO4 cut was fractionated onto a phenyl-Superose HR 5/5 column and enzymatic activity was eluted with a inverse gradient of (NH4)2SO4. Following removal of (NH4)2SO4 with PD-10 columns and fractionation with a Mono Q HR 5/5 column, a sharp peak of enzyme activity was eluted. Analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major band at 106 kDa and, on isoelectrofocusing gel, a pI of 5.1. The activity was completely inhibited by CN- and N3-, suggestive of copper as a likely cofactor. Identification of the protein was confirmed by affinity labeling with 14CN- and isoelectrofocusing. The "amadoriase" activity was also inhibited by Hg2+, Ag2+, Cu2+, and Zn2+ and had Km and Vmax values of 0.14 mM and 0.48 unit/ml (16 units/mg of protein), respectively, for epsilon-(1-deoxyfructosyl) aminocaproate. Significant activity was noted toward many glycated amino acids (highest with epsilon-fructosyl lysine) but not with glycated proteins. The sequence of the first 16 NH2-terminal amino acids and a search in various data bases revealed that this amadoriase enzyme is a novel protein. Based on its properties, this deglycating enzyme, which degrades Amadori products oxidatively into free fructosamine, is classified as fructosyl aminocaproate:oxygen oxidoreductase (EC 1.5.3).
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PMID:Purification and characterization of a membrane-bound deglycating enzyme (1-deoxyfructosyl alkyl amino acid oxidase, EC 1.5.3) from a Pseudomonas sp. soil strain. 895 17

Dermatan sulphates have been shown to inhibit thrombus formation and thrombus growth in different experimental model of venous thrombosis. At variance with heparins, they show a remarkably low haemorrhagic potential. On the other hand, very few data are available on the effect of dermatan sulphates on arterial thrombus formation. We evaluated the effects of a low molecular weight (LMW)-dermatan sulphate, a high molecular weight (HMW)-dermatan sulphate and sulodexide (a mixture of fast-moving heparin fraction and dermatan sulphate) in comparison with LMW- and HMW-heparin, in a model of arterial thrombosis in rats. The insertion of an artificial prosthesis into the abdominal aorta of the animals induced the formation of an occluding thrombus within 2-5 days. The time in which 50% of the loops became occluded (OT50) was also calculated and used to compare the efficacy of the different drug treatments. This was 2.84 days for control animals and 4.25 and 5.80 days for HMW- and LMW-dermatan sulphate, respectively. Neither drug changed the "template" bleeding time, even at higher doses. In contrast, HMW-heparin at doses (8 mg/kg, sc, twice a day) inducing an antithrombotic activity comparable to that of dermatan sulphates, dramatically prolonged the bleeding time. LMW-heparin at the same doses was ineffective. Sulodexide (10 mg/Kg, sc, twice a day) prolonged the occlusion time to the same extent as HMW-heparin (OT50 5.10 vs. 4.14 days), with less an effect on the bleeding time (144 +/- 6 s vs. > 300 s, respectively). Histological examination confirms that the prolongation of occlusion time induced by the drugs is really related to thrombus formation inhibition at the site of arterial wall injury. Acetyl salicylic acid (ASA) (100 mg/kg/day in drinking water as lysine acetylsalicylate) did not modify the effect of Desmin 370 and Sulodexide on both occlusion and bleeding time. However, while it did not increase the antithrombotic activity of HMW-heparin, it significantly prolonged its haemorrhagic effect. In conclusion, dermatan sulphates are effective inhibitors of arterial thrombosis in rats, without inducing bleeding complications.
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PMID:Antithrombotic activity of dermatan sulphates, heparins and their combination in an animal model of arterial thrombosis. 897 38

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein monooxygenase that catalyzes the incorporation of one atom of molecular oxygen into p-hydroxybenzoate to form 3,4-dihydroxybenzoate. The enzyme activates the substrate at the 3 position to electrophilic substitution by lowering the pKa of the phenolic oxygen. The results presented here indicate that regions of positive potential in the active site facilitate this substrate activation, which is necessary for rapid hydroxylation. We have neutralized a positive point charge by mutating lysine 297 to methionine (K297M). This mutation changes an amino acid near the active site, but not directly in contact with the flavin or the substrate. A variety of transient state kinetic and static parameters have been determined with two substrates. The results indicate that the K297M mutant does not activate the substrate through phenolic ionization to the same extent as wild-type (WT) and yet remains a competent hydroxylase. However, catalysis by the mutant is slow compared to that of WT, particularly in the oxidative half-reaction. Thus, normally quite labile oxygenated flavin intermediates encountered in the hydroxylation pathway of WT p-hydroxybenzoate hydroxylase are stabilized and their decay is rate limiting in the K297M turnover. Electrostatic potential calculations offer an explanation for the lack of substrate activation. The stability of the oxidative reaction intermediates seems to be related to a lower degree of substrate activation.
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PMID:Electrostatic effects on substrate activation in para-hydroxybenzoate hydroxylase: studies of the mutant lysine 297 methionine. 920 Jul 6

Nepsilon-(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, approximately 0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n = 22, p >> 0.5). Levels of CML in erythrocyte membrane proteins were approximately 0.2 mmol/mol of lysine for both control and diabetic subjects (p >> 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p = 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.
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PMID:Carboxymethylethanolamine, a biomarker of phospholipid modification during the maillard reaction in vivo. 921 92

The interaction of various small aliphatic and aromatic ionic ligands with the human plasminogen (HPg) recombinant kringle 2 (r-K2) domain has been investigated by 1H-NMR spectroscopy at 500 MHz. The results are compared against ligand-binding properties of the homologous, lysine-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The investigated ligands include the omega-aminocarboxylic acids 4-aminobutyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic acid (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivatives with free and blocked alpha-amino and/or carboxylate groups, and a number of cyclic analogs, zwitterions of similar size such as trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA), and the nonzwitterions benzylamine and benzamidine. Equilibrium association constant (Ka) values were determined from 1H-NMR ligand titration profiles. Among the aliphatic linear ligands, 5-APA (Ka approximately 3.4 mM-1) shows the strongest interaction with r-K2 followed by 6-AHA (Ka approximately 2.3 mM-1), 7-AHA (Ka approximately 0.45 mM-1), and 4-ABA (Ka approximately 0.22 mM-1). In contrast, r-K1, K4, and K5 exhibit a preference for 6-AHA (Ka approximately 74.2, 21.0, and 10.6 mM-1, respectively), a ligand approximately 1.14 A longer than 5-APA. Mutations R220G and E221D increase the affinity of r-K2 for these ligands but leave the selectivity profile essentially unaffected: 5-APA > 6-AHA > 7-AHA > 4-ABA (Ka approximately 6.5, 3.9, 1.8, and 0.74 mM-1, respectively). We find that, while r-K2 definitely interacts with Nalpha-acetyl-L-lysine and L-lysine (Ka approximately 0.96 and 0.68 mM-1, respectively), the affinity for analogs carrying a blocked carboxylate group is relatively weak (Ka approximately 0.1 mM-1). We also investigated the interaction of r-K2 with L-arginine (Ka approximately 0.31 mM-1) and its derivatives Nalpha-acetyl-L-arginine (Ka approximately 0.55 mM-1), Nalpha-acetyl-L-arginine methyl ester (Ka approximately 0.07 mM-1), and L-arginine methyl ester (Ka approximately 0.03 mM-1). Zwitterionic gamma-guanidinobutyric acid, containing one less methylene group than arginine, exhibits a Ka of approximately 0.28 mM-1. The affinity of r-K2 for lysine and arginine derivatives suggests that K2 could play a role in intermolecular as well as intramolecular interactions of HPg. As is the case for the HPg K1, K4, and K5, among the tested ligands, AMCHA is the one which interacts most firmly with r-K2 (Ka approximately 7.3 mM-1) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit Ka values of approximately 4.0, approximately 0.04, and approximately 0.03 mM-1, respectively. The relative stability of these interactions indicates a strict requirement for both cationic and anionic polar groups in the ligand, whereas the presence of a lipophilic aromatic group seems to be of lesser consequence. Ligand-induced shifts of r-K2 (1)H-NMR signals and two-dimensional nuclear Overhauser effect (NOESY) experiments in the presence of 6-AHA reveal direct involvement of residues Tyr36, Trp62, Phe64, and Trp72 (kringle residue numbering convention) in ligand binding. Starting from the X-ray crystallographic structure of HPg K4 and the intermolecular 1H-NMR NOE data, two models of the K2 lysine binding site complexed to 6-AHA have been derived which differ mainly in the extent of electrostatic pairing between the K2 Arg56 and Glu57 side chains. Competition between these two conformations in equilibrium may account for the relatively lesser affinity of the K2 domain for zwitterionic lysine-type ligands.
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PMID:Ligand preferences of kringle 2 and homologous domains of human plasminogen: canvassing weak, intermediate, and high-affinity binding sites by 1H-NMR. 930 49

An NMR method is described for determining residue-specific acid dissociation constants for peptides which contain more than one residue of the same acidic or basic amino acid. The method is based on using the differences in C alpha H proton chemical shifts which result from peptide sequence nearest-neighbor and possibly secondary structure effects to resolve resonances for carbon-bonded reporter protons adjacent to each side-chain acidic group in two-dimensional total correlation spectroscopy (TOC-SY) spectra. Acid dissociation constants were determined for each of the four lysine side-chain ammonium groups of the peptide Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2. Resonances for the C epsilon H2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the C alpha H-C epsilon H2 cross peaks in the TOCSY spectrum. Chemical shift-pH titration data were obtained for each lysine side-chain ammonium group from one-dimensional subspectra taken from two-dimensional TOCSY spectra measured as a function of pH. The pKAs of the Lys1, Lys5, Lys13, and Lys14 side-chain ammonium groups were determined to be 11.14 +/- 0.01, 10.95 +/- 0.01, 10.96 +/- 0.02, and 11.09 +/- 0.02, respectively. The chemical shift-pH titration data were also analyzed by a pH-independent procedure to obtain relative acid dissociation constants: KA(Lys1)/KA(Lys5) = 0.663 +/- 0.009, KA(Lys1)/KA(Lys13) = 0.703 +/- 0.014, and KA(Lys1)/KA(Lys14) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys1, Lys5, Lys13, and Lys14 of 1:1.508:1.422:1.099. To further demonstrate the utility of this method, acid dissociation constants were determined for the six acidic groups of the peptide Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2. Chemical shift-pH titration curves were obtained for the C beta H2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH. The pKAs of the CyS3 and Cys13 thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively. The relative acid dissociation constants (KA(Cys3)/ (KA(Cys13)) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.
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PMID:Determination of acid dissociation constants of peptide side-chain functional groups by two-dimensional NMR. 936 Apr 89

Extracellular signal-regulated kinases (ERKs) phosphorylate and regulate cytoskeletal components of contractile cells and have been implicated in integrin-mediated adhesion. In this study, we examined the contributions of adherence, cell flattening, and cytoskeletal reorganization to adhesion-induced ERK activation in cultured bovine tracheal myocytes. We found, as evidenced by a reduction in electrophoretic mobility, that adhesion to fibronectin induced phosphorylation of both p44ERK1 and p42ERK2. In-gel kinase assays confirmed activation of both p44ERK1 and p42ERK2 in fibronectin-adherent cells, consistent with the notion that ligand-integrin binding is required for adhesion-induced ERK activation. However, ERK activation was maximal 2-4 h after plating, and adherence to either polystyrene or poly-L-lysine also caused ERK activation (fold increase 4 h after plating: fibronectin, 3.75 +/- 0.33; polystyrene, 3.95 +/- 0.78; poly-L-lysine, 2.14 +/- 0.36). Inspection of myocytes following passage onto fibronectin showed near 100% adhesion and cell spreading after 4 h, whereas cells plated onto poly-L-lysine demonstrated adherence but minimal spreading. To test whether the cytoskeletal reorganization accompanying cell spreading is required for adhesion-induced ERK activation, we assessed ERK activity following pretreatment with cytochalasin D, an inhibitor of actin polymerization. Cytochalasin inhibited both cell spreading and ERK activation following adhesion to fibronectin, but had no effect on growth factor-induced ERK activation in adherent cells. We conclude that adhesion-induced ERK activation in bovine tracheal myocytes may occur independently of ligand-integrin binding and is primarily related to the cell spreading that follows adhesion.
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PMID:Adhesion of bovine airway smooth muscle cells activates extracellular signal-regulated kinases. 937 20

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.
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PMID:Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors. 952 39

The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. Gamma-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.
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PMID:Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase. 954 96


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