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)

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amido[17alpha-3H]estradiol photoreagents (6alpha- and 6beta-ANBA-[17alpha-3H]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents. In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([3H]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue. Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled. This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain. Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6alpha- or 6beta-ANBA-estradiol nitrene derivatives to the peptide.
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PMID:Specific photoaffinity labeling of Tyr-49 on the light chain in the steroid-combining site of a mouse monoclonal anti-estradiol antibody using two epimeric 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amidoestradiol photoreagents. 920 30

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.
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PMID:CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein. 938 68

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.
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PMID:A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase. 963 2

Purified rabbit and sheep sex hormone-binding globulins (SHBGs) were photolabeled by Delta 6-testosterone. The maximal levels of specific incorporation were respectively 0.33 and 0.30 mol of label/mol of homodimer. Tryptic cleavage of photolabeled SHBGs gave a single radioactive peptide for rabbit SHBG and two major radioactive peptides S1 and S2 for sheep SHBG. Edman sequencing of the photolabeled peptide of rabbit SHBG revealed a single sequence corresponding to peptidic fragment Leu-118-Lys-134. Subcleavage of this peptide with elastase led to a single radioactive peptidic fragment corresponding to dipeptide Met-133-Lys-134, identified by mass spectrometry, while deletion of the C-terminal residue with carboxypeptidase B showed that all the radioactivity remained on peptide Leu-118-Met-133, thus demonstrating that photolabeling occurred exclusively on Met-133, the only residue common to the two radioactive subcleaved peptides. Edman sequencing of peptides S1 and S2 of sheep SHBG showed a same single sequence corresponding to residues Gln-126-Arg-140 which contained no identifiable phenylthiohydantoin derivative at cycle 14, thus indicating that in both cases the corresponding Met-139 residue is the main site of photolabeling, as confirmed for peptide S1 by the presence at this cycle of a major peak of radioactivity while in peptide S2 the photoattachment of Delta 6-testosterone was found labile in the conditions of sequencing. The photolabeled peptide S1 was characterized by mass spectrometry which showed the covalent fixation of one mole of Delta 6-testosterone and the presence of a biantennary oligosaccharide attached at Asn-133, which suggests that the steroid-binding site is probably not deeply buried in the SHBG homodimer.
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PMID:Photoaffinity labeling of homologous Met-133 and Met-139 amino acids of rabbit and sheep sex hormone-binding globulins with the unsubstituted Delta 6-testosterone photoreagent. 976 Feb 44

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.
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PMID:A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. 1096 27

Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) demonstrate extensive pigmented nevi in both skin and leptomeninges of the central nervous system resembling human neurocutaneous melanosis. We immunohistochemically detected HGF/SF receptor, Met, in a congenital nevus of an infant with neurocutaneous melanosis, indicating that deregulation of HGF/SF-Met signaling in the critical period of development may lead to this fatal syndrome.
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PMID:Immunohistochemical detection of the c-met proto-oncogene product in the congenital melanocytic nevus of an infant with neurocutaneous melanosis. 1120 33

Sugars and amino acids were removed from potato slices by soaking in water and ethanol. They were then infused with various combinations of sugars (glucose and/or fructose) and amino acids (asparagine, glutamine, leucine, isoleucine, phenylalanine, and/or methionine) and fried. Volatile compounds were trapped onto Tenax prior to gas chromatography-mass spectrometry. Relative amounts of compounds (relative to the internal standard) and relative yields (per mole of amino acid infused into the slices) were determined. Amounts of 10 pyrazines, 4 Strecker aldehydes, and 4 other compounds were monitored. Relative amounts and relative yields of compounds varied according to the composition of the system. For the single amino acid-glucose systems, leucine gave the highest relative amount and relative yield of its Strecker aldehyde. Asparagine and phenylalanine gave the highest total relative amount and total relative yield, respectively, of pyrazines. In the system containing all of the amino acids and glucose, the relative amount of 3-methylbutanal was higher, whereas the amounts of the other monitored Strecker aldehydes were lower. Most of the relative amounts of individual pyrazines were lower compared to the glucose-asparagine system, whereas the total relative yield of pyrazines was lower, compared to all of the single amino acid-glucose mixtures. Addition of fructose to the mixed amino acid-glucose model system generated Strecker aldehydes and pyrazines in ratios that were more similar to those of untreated potato chips than to those from the same system but without fructose. Both the sugars and the amino acids present in potato are crucial to the development of flavor compounds in fried potato slices.
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PMID:Formation of Strecker aldehydes and pyrazines in a fried potato model system. 1151 84

The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described. Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A. The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry. It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality. In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra. The amino termini of both P. pastoris- and S. cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine. The steady-state kinetic properties of P. pastoris-expressed MAO-A are similar to those of S. cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine. Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction. Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite. These data demonstrate significant advantages in the heterologous expression of human MAO-A in P. pastoris compared with the published S. cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme.
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PMID:High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae. 1181 36

In this study, we have analyzed experimentally the helical intrinsic propensities of non-charged and non-aromatic residues at different C-terminal positions (C1, C2, C3) of an Ala-based peptide. The effect was found to be complex, resulting in extra stabilization or destabilization, depending on guest amino acid and position under consideration. Polar (Ser, Thr, Cys, Asn, and Gln) amino acids and Gly were found to have significantly larger helical propensities at several C-terminal positions compared with the alpha-helix center (-1.0 kcal/mole in some cases). Some of the nonpolar residues, especially beta-branched ones (Val and Ile) are significantly more favorable at position C3 (-0.3 to -0.4 kcal/mole), although having minor differences at other C-terminal positions compared with the alpha-helix center. Leu has moderate (-0.1 to -0.2 kcal/mole) stabilization effects at position C2 and C3, whereas being relatively neutral at C1. Finally, Met was found to be unfavorable at C1 and C2 ( +0.2 kcal/mole) and favorable at C3 (-0.2 kcal/mole). Thus, significant differences found between the intrinsic helical propensities at the C-terminal positions and those in the alpha-helix center must be accounted for in helix/coil transition theories and in protein design.
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PMID:Amino acid intrinsic alpha-helical propensities III: positional dependence at several positions of C terminus. 1191 21

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.
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PMID:Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI). 1191 35


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