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)

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative. 295 12

Proton inventories (rate measurements in mixtures of H2O and D2O) were determined for the human leukocyte elastase catalyzed hydrolyses of thiobenzyl esters and p-nitroanilides of the peptides MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The dependencies of k2/Ks on mole fraction of solvent deuterium for the p-nitroanilides are "dome-shaped" and were fit to a model that incorporates the mechanistic features of generalized solvent reorganization when substrate binds to enzyme and partial rate limitation of k2/Ks by physical and chemical steps [Stein, R. L. (1985) J. Am. Chem. Soc. 107, 7768-7769]. The proton inventories for the deacylation of MeOSuc-Val-HLE and MeOSuc-Pro-Val-HLE are linear while those for the deacylation of MeOSuc-Ala-Pro-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE are "bowl-shaped" and could be fit to a quadratic dependence of rate on mole fraction of deuterium. These results are interpreted to suggest that the correct operation of the catalytic triad is dependent on substrate structure. Minimal substrates, which cannot interact with elastase at remote subsites, are hydrolyzed via a mechanism involving simple general-base catalysis by the active site histidine and transfer of a single proton in the rate-limiting transition state. In contrast, tri- and tetrapeptide substrates, which are able to interact at remote subsites, are hydrolyzed by a more complex mechanism of protolytic catalysis involving full functioning of the catalytic triad and transfer of two protons in the rate-limiting transition state. Finally, the proton inventories for the deacylation of MeOSuc-Ala-Pro-Ala-HLE and MeOSuc-Ala-Ala-Pro-Ala-HLE are dome-shaped and suggest that the chemical events of acyl-enzyme hydrolysis are only partially rate limiting for these reactions and that some other physical step is also partially rate limiting.
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PMID:Catalysis by human leukocyte elastase: proton inventory as a mechanistic probe. 303 50

A lysine-reactive cross-linker has been coupled to the minor base 3-(3-amino-3-carboxypropyl)uridine in the variable loop of the Escherichia coli elongator methionine tRNA (tRNA(mMet]. Incubation of the derivatized tRNA with E. coli methionyl-tRNA synthetase (MetRS) resulted in covalent coupling of the protein and nucleic acid and loss of amino acid acceptor activity of the enzyme. One mole of tRNA was cross-linked per mole of enzyme inactivated. Enzyme activity was largely restored by release of the bound tRNA following cleavage of the disulfide bond in the cross-linker with a sulfhydryl reagent. The cross-linking reaction was effectively inhibited by unmodified tRNA(mMet) but not by noncognate tRNA(Phe). The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were isolated by anion-exchange chromatography. The cross-linked peptides were released from the tRNA by cleavage in the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding one major peptide plus several minor peptides. Amino acid analysis indicated that the major product was an octadecapeptide cross-linked to tRNA(mMet) through lysine residue 596 in the primary sequence of MetRS. The N-terminal sequence of the peptide was determined to be Val-Ala-Leu-Ile-Glu-Asn-Ala-Glu-Phe-Val, corresponding to residues 582-591 in MetRS. The procedures described here should be applicable to the determination of peptide sequences near the variable loop of other tRNAs containing the 3-(3-amino-3-carboxypropyl)uracil base when such tRNAs are bound to specific proteins.
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PMID:Covalent coupling of the variable loop of the elongator methionine tRNA to a specific lysine residue in Escherichia coli methionyl-tRNA synthetase. 310 75

Subattomole analysis of fluorescein isothiocyanate (FITC) derivatives of amino acids is accomplished by combining capillary zone electrophoresis for high-efficiency separation with laser-induced fluorescence for high-sensitivity detection. Concentration detection limits range from 5 x 10(-12) molar for alanine to 9 x 10(-11) molar for lysine, injected in the column; 9 x 10(-21) mole of alanine is contained within the approximately 1-nanoliter injection volume at the detection limit. The alanine detection limit corresponds to fewer than 6000 molecules injected onto the column and represents an improvement of four orders of magnitude in the state of the art for fluorescent detection of amino acids and an improvement of six orders of magnitude in the state of the art for the detection limit for isothiocyanate derivatives of amino acids.
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PMID:Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence. 314 Mar 81

An alanine racemase, encoded by the alr (dal) gene and believed to be the biosynthetic source of D-alanine for cell wall formation, was purified to homogeneity from an overproducing strain of Salmonella typhimurium (dadB), and the enzymological properties of this enzyme were compared with those of the dadB alanine racemase that functions in the catabolism of L-alanine [Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., & Walsh, C. T. (1984) Biochemistry 23, 5182]. The alr-encoded enzyme has a monomeric structure with a molecular weight of about 40 000. One mole of pyridoxal 5'-phosphate is bound per mole of enzyme, which is essential for catalytic activity of the enzyme. After the internal Schiff base with pyridoxal 5'-phosphate was reduced with NaB3H4, followed by carboxamidomethylation and tryptic digestion of the enzyme, the amino acid sequence of the pyridoxal 5'-phosphate binding peptide was determined. The sequence of 10 amino acid residues around the lysine residue, to which pyridoxal 5'-phosphate is bound, was identical with that of the dadB racemase. No homology was found in the amino-terminal amino acid sequence between the two enzymes. The enzyme was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and D-O-acetylserine in a mechanism-based fashion with a common partition ratio of about 150. The enzyme was labeled with an equimolar amount of [14C]-D-beta-chloroalanine. The inactivator-pyridoxal 5'-phosphate adduct was isolated and shown to be the same structure formed in the dadB racemase inactivation [Roise, D., Soda, K., Yagi, T., & Walsh, C. (1984) Biochemistry 23, 5195].
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PMID:Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene. 352 77

Human plasma glutathione peroxidase (GPx) was purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-150, chromatography on DEAE Sephacel, chromatofocusing with polybuffer, and gel filtration with Sephadex G-75. This isolation resulted in about 5,400-fold purification of the enzyme with a 32% yield in enzyme activity. The final preparation had a specific activity of about 28 units (mmoles NADPH oxidized) per milligram of protein. Determination of selenium on the purified enzyme revealed a content of 3.8 g atoms per mole GPx. Gel electrophoresis using SDS with standard proteins revealed a molecular weight of about 23,000 for the subunits, which would indicate a molecular weight of about 92,000 for the native enzyme. Amino acid analyses of the purified GPx indicated aspartate, glutamate, proline, glycine, alanine, and leucine as the predominant amino acids and cysteine, methionine, tryptophan, and histidine as the minor amino acids.
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PMID:Properties of glutathione peroxidase isolated from human plasma. 366 26

This study characterizes the substrate metabolism of isolated jejunal epithelial cells. Utilization of substrates was assessed by spectrophotometric assay. Significant quantities of glucose, glutamine, and ketone bodies were consumed in a 1-h period; lactate and ammonia were produced. [U-14C]glucose was metabolized in this medium to approximately three moles of lactate per mole of CO2. The pattern of tricarboxylic acid (TCA) cycle metabolism was analyzed utilizing media containing different concentrations of potential metabolic substrates and trace quantities of [14C]- succinate. O2 consumption rates indicated that glutamine can serve as an energy source in the absence of other substrates. Relative 14CO2 production from [1,4-14C]succinate versus [2,3-14C]succinate, which estimates flux of TCA cycle intermediates to products other than CO2, was increased more than twofold when glutamine was the only major substrate available. Alanine was produced from TCA cycle intermediates. Analysis of the citrate labeling pattern in the presence of [2,3-14C] succinate suggested that carbon from the TCA cycle does not form a significant fraction of acetyl-CoA used for citrate synthesis and that glutamine carbon was not completely oxidized to CO2. These findings suggest that glucose and glutamine are converted to three-carbon compounds by the jejunal epithelium.
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PMID:Substrate metabolism of isolated jejunal epithelium: conservation of three-carbon units. 395 76

Eggs of Urechis caupo are surrounded by a congruent to 0.9 micrometer thick egg envelope and, attached to that, a peripheral jelly layer about 3 micrometers thick. Before fertilization, the sperm undergoes the acrosome reaction and binds to the egg envelope. As part of a study of the induction of the acrosome reaction and sperm binding in Urechis, we have developed a method to prepare an egg envelope fraction by differential centrifugation. The isolation procedure removes much of the jelly layer, but does not alter the fine structure of the envelope. When a sperm contacts an isolated envelope, it undergoes a normal acrosome reaction and binds to the envelope's outer face. Electrophoresis of the envelope fraction on sodium dodecyl sulphate (SDS)/polyacrylamide gels revealed six major components stained by Coomassie Blue, of which four are stained by the periodic acid-Schiff reagents (PAS). To measure the degree of enrichment of the envelope fraction, envelopes were isolated from eggs that had been externally radio-iodinated; the specific activity of the envelope fraction was 17 +/- 3 times greater than that of intact eggs. The amino acid composition of the envelope fraction is dominated by Gly (19 mole %), Asx (11%), Thr (11%), Ser (8%), Ala (8%) and Glx (8%). The sugars fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine were detected by gas-liquid chromatography. We also investigated whether the egg envelope changes at fertilization. No change was detected in the electrophoretic 125I pattern of externally radio-iodinated eggs, and the envelope fractions prepared from unfertilized and fertilized eggs produced the same Coomassie Blue pattern on SDS/polyacrylamide gels.
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PMID:Isolation and partial characterization of Urechis caupo egg envelopes. 404 Sep 20

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.
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PMID:Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria. 406 97

The thiopeptins are a new group of sulfur-containing peptide antibiotics produced by Streptomyces tateyamensis. The antibiotic consists of a major component (designated as thiopeptin B) and four minor ones (thiopeptins A(1) to A(4)). These components were isolated by solvent extraction from mycelium followed by chromatography on silica gel with various ratios of chloroform and methanol as elution solvents. Acid hydrolysis of each of the thiopeptin components yielded 1 mole of valine, 1 of threonine, 1 of cysteine, and 2 of alanine as amino acids. Each component of the thiopeptin A group has chemical and biological properties closely similar to those of thiopeptin B, but detailed characterization has established that thiopeptins A(1), A(3), and A(4) are new antibiotics. We could not obtain accurate data for determination of the uniqueness of A(2) because of insufficient sample. Thiopeptin has strong antibacterial activity against gram-positive bacteria and Mycoplasma, and exhibits no cross-resistance to major human-use antibiotics.
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PMID:Thiopeptin, a new feed additive antibiotic: microbiological and chemical studies. 504 67


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