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)

At substrate concentrations, in medium, of 0.2 to 20 mM and at temperatures of 25 and 37 degrees C, the initial concentrative influx of the amino acids L-lysine (30 and 37 degrees C), L-valine, and gamma-aminobutyric acid into incubated mouse-cerebrum slices follows the rate equation for the initial influx of alpha-aminoisobutyric acid (Cohen, J. Physiol. 228:105, 1973), v equals Vmax/(1+Kt/S)+kuS. Kinetic constants at 37 degrees C are: Vmax equals 0.089 mumoles/g final wet wt of slices, min, Kt equals 0.69 mM, ku equals 0.037 mumoles/g final wet wt, mM-substrate, min for L-lysine; Vmax equals 0.60, Kt equals 1.30, ku equals 0.067 for L-valine; and Vmax equals 1.71, Kt equals 1.58, ku equals 0.094 for gamma-aminobutyric acid. The linear term, kuS, is due to an unsaturable process of concentrative uptake, not diffusion. Comparison of temperature coefficients reveals a "reference" pattern for typical low affinity transport of amino acids into brain slices. Its characteristics are: Activation energies associated with Vmax and ku are in range 14 to 20 kcal/mole; K, varies only slightly with temperature, L-Lysine and alpha-aminoisobutyric acid fit this pattern; L-valine and gamma-aminobutyric acid deviate in part. The Akedo-Christensen plot (J. Biol. Chem. 237:118, 1962) does not distinguish between the rateequation v equals Vmax/(1+Kt/S)+kuS for saturable uptake plus first-order unsaturable concentrative uptake, and the rate equation v equals Vmax/(1 + Kt/S)+kd(S minus Si) for saturable uptake plus first-order nonconcentrative "passive diffusion".
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PMID:A comparison of the rate equations, kinetic parameters, and activation energies for the initial uptake of L-lysine, L-valine, gamma-aminobutyric acid, and alpha-aminoisobutyric acid by mouse brain slices. 112 85

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.
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PMID:Inactivation and covalent modification of CTP synthetase by thiourea dioxide. 130 49

The bifunctional reagent 1,4-dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min-1 M-1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme-bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 microM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit. Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn-Ile-X1-Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn-Ile-X1-Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn-Ile-X1-Lys cross-linked to X2-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross-linked peptides but not with formation of Asn-Ile-X1-Lys, inactivation is likely caused by the reaction leading to the cross-link between Cys151 and Cys164. The distance between the alpha-carbons of these residues in the crystal structure is 15.5 A, whereas only 12.0 A can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase undergoes a conformational change in forming the cross-link or that local rapid fluctuations in structure occur in solution to the extent of 3.5 A in this region of pyruvate kinase.
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PMID:Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase. 130 66

The NCI-H69 cell alpha 1----3fucosyltransferase has been purified from a 0.2% Triton X-100R solubilized enzyme fraction by GDP-hexanolamine-Sepharose affinity chromatography and Superose 12 gel filtration. Photoaffinity labeling experiments with 125I-GDP-hexanolaminyl-4-azidosalicylic acid present in concentrations equivalent to 0.5 and 1 times Ki of the inhibitor for the enzyme indicated that labeling of the 45-kDa protein band could be inhibited by addition of 400 microM GDP-fucose but was not effected by similar concentrations of either GDP-mannose or GDP-glucose. The purified enzyme was applied to studies intended to define catalytically essential amino acid residues of the protein. Incubation of the enzyme in the presence of increasing concentrations of pyridoxal 5'-phosphate was found to result in irreversible inactivation of the enzyme after NaBH4 reduction. The donor substrate, GDP-fucose, was found to protect the enzyme from inactivation. Little or no protection was found for either GDP-mannose or the acceptor substrate nLc4. Pyridoxal 5'-phosphate was shown to behave as a competitive inhibitor with respect to GDP-fucose with a Ki of 105 microM. Labeling with 3H-pyridoxal 5'-phosphate resulted in the incorporation of approximately 8 mol pyridoxal 5'-phosphate per mole subunit. Parallel experiments containing GDP-fucose indicated protection of one site per subunit correlated with GDP-fucose binding. Acid hydrolysis and chromatographic analysis of the 3H-pyridoxylated protein indicated greater than 95% of the 3H label was recovered as pyridoxyl-lysine irrespective of whether GDP-fucose was present or not during labeling. These studies indicate the presence of a catalytically essential lysine residue associated with GDP-fucose binding to this enzyme. This information will be of value in further studies of this and other alpha 1----3fucosyltransferases and may suggest a practical basis for modulation of enzyme activity in the cell.
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PMID:Presence of an essential lysine residue in a GDP-fucose protected site of the alpha 1----3fucosyltransferase from human small cell lung carcinoma NCl-H69 cells. 132 90

The amino acid composition of flavokinase has been determined to contain five arginyl and four lysyl residues per mole. Flavokinase is inactivated by arginine-specific reagents. The substrates riboflavin and especially ATP impede inactivation, whereas neither of the products, ADP or FMN, protect. Among lysine-modifying reagents, only 2,4,6-trinitrobenzene sulfonic acid caused inactivation of the enzyme, especially under conditions for denaturation. In this case it was noted that the activity was enhanced at the early stage of the reaction but this enhancement was repressed in the presence of ATP along with the significant protection of activity. Results with modification of arginyl residues and incorporation of 14C-phenylglyoxal suggest that one such residue is involved in the substrate-binding site. The involvement of lysyl residues in catalytic function remains unclear, but seems less critical.
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PMID:Modification of arginyl and lysyl residues of flavokinase from rat small intestine. 133 80

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.
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PMID:Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by o-phthalaldehyde. 141 88

Modification of A. conoides beta-glucosidase by diethylpyrocarbonate caused rapid inactivation of the enzyme. The kinetic analyses showed that the inactivation by diethylpyrocarbonate resulted from the modification of an average of one histidine residue per mole of enzyme. The modified enzyme showed an increase in absorbance at 240 nm. Sulphydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. The substrate offered significant protection against diethylpyrocarbonates modification. The results indicate that diethylpyrocarbonate was interacting with the enzyme at or near the active site.
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PMID:Inactivation of a beta-glucosidase from Arthrobotrys conoides by diethyl pyrocarbonate: evidence of histidine at the active site. 152 73

A potent and structurally novel antimicrobial peptide was purified from the cytoplasmic granules of bovine neutrophils. Suspensions of Staphylococcus aureus and Escherichia coli were virtually sterilized by the peptide at a concentration of 10 micrograms/ml. The peptide was found to be comprised of 13 amino acids, 5 of which were tryptophan residues, and the carboxyl-terminal arginine was carboxamidated. The primary structure of the peptide, which we have named indolicidin, is H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2. The mole percent of tryptophan in indolicidin is the highest observed among known protein sequences. The multiple tryptophan residues presumably play an important role in the function of this unique antibiotic peptide.
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PMID:Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. 153 21

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

In an attempt to improve bifunctional chelate labelling of Mab, we investigated the use of a polyamino acid backbone for multiple DTPA substitutions. Poly-L-lysine (PL) (3.8 Kd, n = 25) was partially acetylated with MADTPA to yield 11 moles of DPTA per mole of PL. The average numbers of DTPA on PL were directly quantified with MADTPA-C-14. The remaining epsilon amino groups on PL-DTPA (I) were measured with TNBS reagent. A selective maleimide derivatization of (I) with S-SMPB yielded (II), which contains 2.3 moles of maleimide groups per mole of (I). The sulfhydryl activation of Mab-TP41.2F(ab')2 with 2-Iminothiolane hydrochloride produced (III), containing 1.3 moles of sulfhydryl groups per mole of Mab. Compounds (II) and (III) were combined to form a single thioether-spaced chain linkage of Mab-PL-DTPA (IV), which was subsequently chelated with 111In to yield (V), which was the compound of interest. Indium-111-PL-DTPA (VI) and 111In-DTPA-MabTP41.2F(ab')2 (VII) also were prepared for control studies. Direct cell binding assay revealed the mean immunoreactivity of (V) to be 79.4% and that of (VII) to be 39.5%. In a biodistribution study on melanoma tumor-bearing athymic mice at 4, 24, and 48 hr postinjection, the tumor/blood and tumor/liver ratios at 48 hr were 11.6 and 1.2 for (V), compared to 3.7 and 0.13, respectively, with (VII). Thus, the PL configuration for radiolabeled antibodies seems to result in decreased hepatic accumulation and retained tumor activity. The findings suggest that further studies of this new compound are warranted.
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PMID:Reduced hepatic accumulation of radiolabeled monoclonal antibodies with indium-111-thioether-poly-L-lysine-DTPA-monoclonal antibody-TP41.2F(ab')2. 155 42


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