Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously reported that an extract of uremic plasma reduces binding of phenytoin and tryptophan by normal plasma and plasma albumin. This effect appears to reproduce the impaired binding of many drugs and several endogenous metabolites by uremic plasma. In the present study we further characterized the properties of extracts from uremic sera and body fluids using binding of salicylate as a model. Salicylate was chosen because it binds to both of the main albumin binding loci for aromatic, acidic drugs. Using a computer-assisted, least-squares, curve-fitting program, LIGAND, we found that the most satisfactory model for salicylate binding to 1:10 diluted normal plasma was a binding number (n) of 2 mol of salicylate per mole of albumin with an association constant (k) of 2.85 X 10(4) L/mol, an additional binding of 0.5 mol to other sites on albumin or to other proteins, and nonspecific binding of 21%. Addition of uremic pleural fluid extract to diluted normal plasma produced a monotonic decline in k to 0.17 X 10(4) L/mol with no change in n except possibly at the highest dose of uremic inhibitor. This pattern of competitive inhibition indicates presence of unknown ligands in the uremic extract that compete at both binding loci. More efficient extraction methods might also yield additional ligand(s) that inhibit through a noncompetitive mechanism.
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PMID:Inhibition of salicylate binding to normal plasma by extracts of uremic fluids. 647 46

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase. 650 17

Interaction of N-bromosuccinimide with the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex results in a rapid and specific modification of two tryptophan residues per mole of the protein and in complete inactivation of the enzyme. Modification of the enzyme excludes the development of the negative Cotton effect with a maximum at 330 nm, characteristic of the charge transfer complex between the protein tryptophan residue and thiamine pyrophosphate. Modification of one tryptophan residue was shown to result in the absence of the band at 330 nm in one of the active centers of pyruvate dehydrogenase, while oxidation of two tryptophan residues excludes the formation of a charge transfer complex in both centers. The conclusion was drawn about the presence in the pyruvate dehydrogenase active centers of two tryptophan residues involved in the formation of the charge transfer complex with the thiazolium ring of thiamine pyrophosphate.
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PMID:Localization of tryptophan residues in thiamine pyrophosphate-binding sites of pyruvate dehydrogenase from pigeon breast muscle. 651 55

At pH 4.1, bovine thrombin reacts rapidly with N-bromo-succinimide to yield modified enzyme containing oxidized tryptophan residue. Both fibrinogen clotting activity and esterase activity are reduced considerably when three moles of tryptophan residues per mole of thrombin are oxidized, but the Michaelis constants for synthetic substrates are not appreciably altered. Reaction of NBS also results in a decrease in the affinity of thrombin for heparin. The dissociation constant for heparin-thrombin complex is increased by 2.6-fold due to the modification of one tryptophan residue. However, the magnitude of the increase in the dissociation constant remains the same for modified enzymes containing approximately two or three oxidized tryptophan residues. The rate constant for the inactivation of thrombin by antithrombin III is increased by 2.5-fold due to the modification of a single tryptophan residue. This increase in rate constant is not further amplified when more than one tryptophan residue is oxidized. In contrast, in the presence of heparin the rate of inactivation of modified and unmodified thrombins by antithrombin III are not significantly different. Thus, the heparin-sensitized inactivation of thrombin by antithrombin III is affected by the modification of one tryptophan residue. Spectrophotometric titrations of the phenolic hydroxyl groups suggest that the structural environments of tyrosyl groups for both unmodified and modified thrombin containing one oxidized tryptophan residue, are similar. The temperature for half loss of catalytic activity of control and NBS-modified thrombin, containing one oxidized tryptophan, are 52 and 51.5 degrees C respectively. It appears that the one tryptophan residue of thrombin is situated at or close to the binding site of heparin.
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PMID:Catalytic and regulatory functions of N-bromosuccinimide-modified bovine thrombin. 652 42

The major gamma-carboxyglutamic acid-containing protein of rabbit cortical bone isolated and purified from near-neutral (pH 7.5) EDTA-extracts by DEAE-cellulose and Sephadex G 75 column chromatography had a molecular weight of about 5600 based on integral amino-acid composition; this was confirmed by high-performance liquid chromatography. The purified protein had high glutamic acid and aspartic-acid contents, two to three residues of gamma-carboxyglutamic acid per molecule and zero levels of serine, threonine, methionine, histidine and tryptophan. Equilibrium dialysis indicated that the protein has a weak affinity for calcium ions with a formation constant of 1515 M-1 at I 0.15, pH 7.5, 25 degrees C with a binding capacity of 2 mol calcium per mole protein.
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PMID:The small molecular weight, gamma-carboxyglutamic acid-containing protein of rabbit bone tissue. 659 60

The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis, 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E1%1cm) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of lecithin-cholesterol acyltransferase from human plasma. 3. Chemical properties of the enzyme. 662 99

The semireduced, semioxidized, and OH(.)-adduct radicals of bilirubin (BR) and biliverdin (BV) have been characterized using pulse radiolysis techniques. Laser flash photolysis (265-nm) of these pigments led to monophotonic photoionization with quantum yields of 0.08 for BR and 0.03 for BV. No evidence for triplet formation or for photoisomerization was found after 265-nm laser excitation. However, 347-nm excitation of BR in chloroform led to simultaneous photoisomerization and radical formation, but the radicals are thought to have originated from a pathway other than photoionization. The relevance of these observations to BR photoreactivity is discussed. BR radical ions in alkaline solution did not react with tryptophan (TrpH), but the semioxidized TrpH radical oxidized BR with k = 4.3 X 10(8) dm3 mole-1 sec-1. When human serum albumin (HSA) was oxidized using radiolytically generated azide radicals, a radical transformation involving TrpH and TyrOH residues occurred with k = 3.8 X 10(3) sec-1. When BR was complexed with the protein the transformation rate was reduced to 1.6 X 10(3) sec-1. This was interpreted in terms of a conformational change in the protein. Identification of the probable residues involved provided information about the primary BR binding site which was consistent with an earlier report.
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PMID:The radical ions and photoionization of bile pigments. 665 16

A model system was developed to (a) reflect the chemical attributes of the microenvironment involved in albumin-eicosanoid interactions and (b) determine the effects of other ligands on these interactions. Albumin-dependent modulation of prostaglandin stability was chosen as the basis for this system. 15-Ketoprostaglandin E2 (PGE2) was evaluated as a model ligand because under special conditions it decomposes with formation of a visible chromophore. Human serum albumin, in a concentration-dependent fashion, catalyzed the dehydration of 15-keto-PGE2 with the concurrent generation of this chromophore (lambda max = 505 nm, epsilon = 35,000). Since chromophore production from 15-keto-PGE2 in albumin-free solution occurs only at pH greater than 10, the results suggest that albumin-eicosanoid interactions involve a microenvironment with alkaline attributes. The effect of other ligands on albumin-15-keto-prostaglandin E2 interactions was determined by monitoring their ability to inhibit the spectral component of these interactions. Inhibition correlated with an affinity for specific binding sites on albumin. At mole ratios of ligand/albumin below 1, only phenylbutazone, its analogs, and warfarin inhibited chromophore development. Other ligands including fatty acids, steroids, tryptophan, and drugs with an affinity for other binding sites were ineffective inhibitors.
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PMID:Albumin-eicosanoid interactions. A model system to determine their attributes and inhibition. 669 91

We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The interaction of aromatic amino acids with rat liver phenylalanine hydroxylase. 670 37

1. A low molecular weight (7,700) Zn- and Cu-containing protein was isolated from the livers of Zn-injected rainbow trout by gel filtration and ion exchange chromatography. Purity of the isolated protein was assessed by native and denaturing polyacrylamide gel electrophoresis and isoelectric focusing. 2. The purified protein was positively identified as a metallothionein on the basis of its molecular size, high metal content (3.6 g atoms Zn and 2.6 g atoms Cu per mole; 5.2% metal), heat stability, u.v. absorption spectrum, charge and amino acid composition (25% cysteine, no histidine and tryptophan, and trace tyrosine and phenylalanine). 3. The relatively high Cu content of this protein was unexpected and may be attributed to the presence of high levels of Cu, as Cu-thionein, in the livers of non-injected fish. 4. The comparative differences in the metal content of hepatic MT in trout and other animals are discussed.
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PMID:Isolation and characterization of hepatic metallothionein from rainbow trout (Salmo gairdneri). 683 17


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