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

Mitochondrial malate dehydrogenase (MDH) was found to be rapidly inactivated by o-phthalaldehyde. MDH-o-phthalaldehyde adduct gives a characteristic absorption maximum at 337 nm. Moreover, this derivative shows fluorescence emission maxima at 405 nm when excited at 337 nm and 280 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The enzyme was found to be protected against o-phthalaldehyde inactivation by NADH, indicating that the essential residues are present at or near the coenzyme binding site. Stoichiometric results indicate that 4 isoindole derivatives are formed per enzyme molecule upon complete inactivation. However, 90% of the activity loss was accompanied by the formation of 2 moles of isoindole per mole of the enzyme. These approaches give consistent evidence that two cysteines along with two lysines in close proximity are essential for the enzymatic activity.
 ...
PMID:Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by o-phthalaldehyde. 141 88

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.
 ...
PMID:Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method. 153 1

The relationship between derivatization of reactive cysteine residues with N-ethylmaleimide and a partial desensitization of fructose 1,6-bisphosphatase to AMP inhibition was studied. AMP desensitization of the enzyme was found to be dependent on the activity assay conditions used. When the assay was performed in the presence of high levels of monovalent cations (150 mM), the AMP affinity of the enzyme decreased with the chemical modification. The apparent loss of sensitivity toward AMP was accompanied by an uptake of 1 mole of N-ethylmaleimide/mole of enzyme subunit. However, the modified enzyme did not show alteration in AMP inhibition in the absence of K+. Evidence was obtained that K+ induces a conformational change on the enzyme derivative, which hinders AMP interaction with the protein. The results point to the importance of selecting suitable conditions for the study of the regulatory properties in allosteric enzymes.
 ...
PMID:The interaction of monovalent cations with fructose 1,6-bisphosphatase modified by N-ethylmaleimide and its relation with AMP inhibition. 155 47

The interaction of gold(I) thiomalate (Autm) (Myocrysine) with ergothionine (ErSH) has been studied in aqueous solution at pH 7.4. It was found that ErSH forms a ternary complex of the type ErS-Au-tm at a 1:1 mole ratio; unlike other thiols (e.g., cysteine and glutathione) it does not eject thiomalate (Htm) as a free ligand. However, in the presence of glutathione (GtSH), both ligands, ErSH as well as Htm, from the ErS-Au-tm complex were freed. The 13C NMR chemical shifts of C-2 resonance of ergothionine in the presence of Autm shifts greater than imidazolidine-2-thione (Imt) and 1,3-Diazinane-2-thione (Diaz), indicating the stronger complexation of ErS-Au-tm compared to Imt-Au-tm and Diaz-Au-tm.
 ...
PMID:The 13C NMR study of the binding of gold(I) thiomalate with ergothionine in aqueous solution. 161 2

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate. 167 73

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.
 ...
PMID:A plant metallothionein produced in E. coli. 176 50

The primary structures of the alpha- and beta-hemoglobin chains of the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta) are presented. Chain separation was performed by carboxymethyl-cellulose chromatography. The peptides, obtained by tryptic digestion of the oxidized chains, were prefractionated by gel chromatography and isolated by reversed-phase HPLC. For sequence analysis gas and liquid phase sequencers were employed. The tenrec hemoglobin consists of one alpha- and two beta-chains the latter occurring in a 1:1 ratio and differing in beta 16 Gly/Cys and beta 118 Phe/Leu. Two external cysteine residues at beta 16 and beta 52 cause reversible polymerization to octamers and most likely irreversible formation of higher polymers. A comparison of the whole chains and certain positions of tenrec hemoglobin with those of Insectivora sensu strictu, Scandentia and Proto- and Metatheria corroborates a long and independent evolution of tenrec and its phylogenetic isolation from the Insectivora s.str. (hedgehog, musk shrew and mole). Replacements at positions involved in heme and subunit interface contacts are discussed. Compared to human hemoglobin the tenrec pigment shows a low intrinsic oxygen affinity as well as lower chloride and temperature sensitivities, a reduced Bohr effect and a strong response to 2,3-DPG. The possible adaptive significance of these properties is discussed in relation to the large diurnal body temperature variations seen in tenrecs.
 ...
PMID:Primary structure and oxygen-binding properties of the hemoglobin from the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta). Evidence for phylogenetic isolation. 179 18

An allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromatography. Phl p V binds IgE from serum of grass-sensitized donors as revealed in immunoelectrophoretic techniques and in SDS-PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS-PAGE treatment purified Phl p V is identified as two IgE-binding components, Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30-kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47-kD fraction corresponds to Phl p Va/Phl p Vb as seen in SDS-PAGE, while the 25-kD component presumably corresponds to a degradation product present in whole pollen extract. The NH2-terminal sequence of Phl p V, corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH2 terminal sequence of other proteins. The amino acid composition, revealing 26 mole % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison, Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.
 ...
PMID:Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V. 186 92

Recombinant rat liver guanidinoacetate methyltransferase is inactivated by glutathione disulfide (GSSG) following pseudo-first-order kinetics. A second-order rate constant of 20.8 M-1 min-1 is obtained at pH 7.5 and 30 degrees C. The inactivation is fully reversed by glutathione (GSH) in a pseudo-first-order fashion with a second-order rate constant of 11.1 M-1 min-1. The rate of inactivation is not affected by S-adenosylmethionine or guanidinoacetate, but complete protection against inactivation is observed in the presence of sinefungin plus guanidinoacetate. At equilibrium in the buffers containing various concentrations of GSH and GSSG, the enzyme shows activities that are dependent on the ratio but not on the total concentration of GSH and GSSG. A hyperbolic relationship is obtained between enzyme activity and [GSH]/[GSSG] ratio. The inactivation by GSSG is associated with the disappearance of approximately 1 mol of sulfhydryl group per mole of enzyme. These results indicate that inactivation of guanidinoacetate methyltransferase by GSSG is the consequence of the formation of a mixed disulfide between a protein thiol and glutathione. The equilibrium constant for the redox reaction, E-SH + GSSG in equilibrium with E-SSG + GSH, obtained from the equilibrium data (1.69) is in good agreement with the value determined as the ratio of second-order rate constants for reactivation and inactivation (1.87). The cysteine residue engaged in the mixed disulfide with glutathione is identified as Cys-15 by peptide analysis after consecutive treatment of the GSSG-inactivated enzyme with N-ethylmaleimide, 2-mercaptoethanol, and [14C]iodoacetate. The GSSG-inactivated enzyme binds S-adenosyl-methionine but not guanidinoacetate in the presence and absence of sinefungin. Native guanidinoacetate methyltransferase binds guanidinoacetate in the presence of sinefungin. The low overall redox equilibrium constant of 1.7-1.9 found for the reaction between guanidinoacetate methyltransferase and GSSG suggests that the activity of the enzyme is not amenable to modulation by the change in intracellular [GSH]/[GSSG] ratio.
 ...
PMID:Reversible inactivation of recombinant rat liver guanidinoacetate methyltransferase by glutathione disulfide. 189 65

The reaction of ozone with a number of biological molecules was found to produce singlet oxygen in high yield. At pH 7.0, the reaction of ozone with an equimolar amount of biological molecule produced the following singlet oxygen yields (mole of singlet oxygen/mole of ozone): cysteine, 0.49 +/- 0.02; methionine, 1.13 +/- 0.11; reduced glutathione, 0.33 +/- 0.02; albumin, 1.00 +/- 0.05; uric acid, 0.64 +/- 0.09; ascorbic acid, 0.96 +/- 0.007; NADPH, 1.07 +/- 0.07; NADH, 0.95 +/- 0.01. Thus, singlet oxygen may be an important intermediate in the biochemical damage caused by ozone.
 ...
PMID:Singlet oxygen production from the reactions of ozone with biological molecules. 202 12


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>