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Showing papers on "Substrate (chemistry) published in 1986"


Journal ArticleDOI
TL;DR: The dissociation constant for hirudin was determined by varying the concentration of hirUDin in the presence of a fixed concentration of thrombin and tripeptidyl p-nitroanilide substrate and was markedly dependent on the ionic strength of the assay.
Abstract: The dissociation constant for hirudin was determined by varying the concentration of hirudin in the presence of a fixed concentration of thrombin and tripeptidyl p-nitroanilide substrate. The estimate of the dissociation constant determined in this manner displayed a dependence on the concentration of substrate which suggested the existence of two binding sites at which the substrate was able to compete with hirudin. A high-affinity site could be correlated with the binding of the substrate at the active site, and the other site had an affinity for the substrate that was 2 orders of magnitude lower. Extrapolation to zero substrate concentration yielded a value of 20 fM for the dissociation constant of hirudin at an ionic strength of 0.125. The dissociation constant for hirudin was markedly dependent on the ionic strength of the assay; it increased 20-fold when the ionic strength was increased from 0.1 to 0.4. This increase in dissociation constant was accompanied by a decrease in the rate with which hirudin associated with thrombin. This rate could be measured with a conventional recording spectrophotometer at higher ionic strength and was found to be independent of the binding of substrate at the active site.

565 citations


Journal ArticleDOI
TL;DR: Detailed studies on the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types have allowed the formulation of some general rules which explain, at least in part, the branching patterns and microheterogeneity of these structures.
Abstract: Detailed studies on the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types have allowed the formulation of some general rules which explain, at least in part, the branching patterns and microheterogeneity of these structures. These rules are discussed under the following headings: competition of two or more enzymes for a common substrate; controls at the level of enzyme substrate specificity (e.g., critical sugar residues which turn enzyme activity on or off, branch specificity, and the role of the polypeptide in the glycoprotein substrate); substrate availability.

538 citations


Journal ArticleDOI
TL;DR: Results indicate that the enzyme oxidizes Mn(II) to Mn( III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds.

460 citations


Journal ArticleDOI
TL;DR: Hydrolases can be used to catalyse the synthesis of condensation products such as β-lactam antibiotics, peptides, oligosaccharides and glycerides, and rational analysis of how yield controlling factors may be changed to obtain optimum yields is used to evaluate whether these biotechnological processes can compete with the chemical methods currently used for the synthesis.

329 citations


Journal ArticleDOI
08 Aug 1986-Science
TL;DR: Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin, which has broad peptidase specificity and contains a largeHydrophobic substrate binding cleft.
Abstract: Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly(166)), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (k(cat)/K(m)) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft ( approximately 160 A(3)), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (k(cat)/K(m)) (up to 5000 times) as a result of steric hindrance.

225 citations


Journal ArticleDOI
TL;DR: A rapid, simple and sensitive assay has been developed for tyrosine-3-monooxygenase, the enzyme catalyzing the rate-limiting step in catecholamine biosynthesis.

204 citations


Journal ArticleDOI
TL;DR: In this paper, a new method was proposed for making titanium nitride (TiN) films at substrate temperatures between about 400 and 700°C, which is versatile with growth rates of up to 0.1 μm s −1 possible.

197 citations


Book ChapterDOI
TL;DR: In this article, the authors studied the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types.
Abstract: Detailed studies on the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types have allowed the formulation of some general rules which explain, at least in part, the branching patterns and microheterogeneity of these structures. These rules are discussed under the following headings: competition of two or more enzymes for a common substrate; controls at the level of enzyme substrate specificity (e.g., critical sugar residues which turn enzyme activity on or off, branch specificity, and the role of the polypeptide in the glycoprotein substrate); substrate availability.

196 citations


Patent
30 Oct 1986
TL;DR: In this paper, an enzyme-labeled binding assay is performed using an elongate strip provided with transverse reagent zones, where the substrate moves slower through the absorbent material than the enzyme-labelled reagent or any compound of the enzyme formed in the assay.
Abstract: A device for performing an enzyme-labelled binding assay comprises an absorbent material and a developing solution, wherein the absorbent material is provided with a plurality of reagent zones including an indicator reagent zone, and is capable of transporting the developing solution by capillary action sequentially through each reagent zone, and wherein the indicator reagent zone includes a reagent capable, directly or indirectly, of immobilising an enzyme-labelled reagent in an amount dependent upon the assay result, characterised in that the developing solution includes a signal producing substrate for the enzyme. The substrate moves slower through the absorbent material than the enzyme-labelled reagent or any compound of the enzyme-labelled reagent formed in the assay. The absorbent material is suitably in the form of an elongate strip provided with transverse reagent zones. The device is useful for performing immunoassays including immunometric assays and dual analyte assays.

185 citations


Patent
10 Nov 1986
TL;DR: In this article, a method of using a redox enzyme and redox substrate to detect conversion to an effective mediator by an assay enzyme label of a compound which is non-mediating under the assay conditions is employed.
Abstract: Electrochemical detection of mediator level is employed in a method of assay using a redox enzyme and redox substrate to detect conversion to an effective mediator by an assay enzyme label of a compound which is non-mediating under the assay conditions.

178 citations


Journal ArticleDOI
TL;DR: The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity.

Journal ArticleDOI
TL;DR: ( 2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol, and the product was unequivocally identified as (+)-(2S,4',4',5'-pentahydroxy-flavanone.
Abstract: (2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.

Journal ArticleDOI
TL;DR: Two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicle, which for chemically pure bilayers occurs best in the gel phase, and at least one of these two steps appears to involve enzyme-enzyme interaction.

Journal ArticleDOI
TL;DR: Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex.

Journal ArticleDOI
TL;DR: In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates, however, considerable differences between the bovines and human enzymes were observed with nucleoside substrates.


Journal ArticleDOI
TL;DR: The trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography and two components appeared to be multiply-phosphorylated, but did not contain NeuAc; the possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed.
Abstract: Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.

Journal ArticleDOI
TL;DR: In this paper, the development of a sensitive electrochemical assay for low levels of hydrogen peroxide is described, based on the enzymic reduction of substrate (H2O2) by peroxidase and subsequent electron transfer from a gold or pyrolytic graphite electrode to the enzyme via a redox mediator.

Patent
20 Jun 1986
TL;DR: In this paper, a non-magnetic recording medium comprising a nonmagnetic substrate having formed thereon a magnetic layer is described, and the magnetic layer comprises 1 to 8 wt % of one or more of rare earth elements selected for among B, Al, Si, P, Ge, Sn, Sb, Se, Te, Pb, Bi, Cu, Ti, V, Cr, Zr, Nb, Mo, W, and Ta.
Abstract: Magnetic recording medium comprising a non-magnetic substrate having formed thereon a magnetic layer. The magnetic layer comprises 1 to 8 wt % of one or more of metals selected for among B, Al, Si, P, Ge, Sn, Sb, Se, Te, Pb, Bi, Cu, Ti, V, Cr, Zr, Nb, Mo, W, and Ta, up to 13 wt % of one or more of rare earth elements selected from among Y, La, Ce, Pr, Nd, Sm, Gd, Tb, and Dy, 3 to 13 wt % of oxygen, and the balance of Co and unavoidable impurities. This magnetic layer may contain up to 22 wt % of Ni. This magnetic recording medium is excellent in both corrosion resistance and magnetic properties.

Journal ArticleDOI
TL;DR: It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicle induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.

Journal ArticleDOI
TL;DR: An improved assay method for cholesterol 7α-hydroxylase which is accurate, sensitive and yet still simple is described, which had more than 10-fold increase in the method than the previous one.

Journal ArticleDOI
TL;DR: In this paper, the surface morphology of cubic SiC single crystals grown on Si(100) oriented substrate was observed and texture-like morphology was observed, which originated from antiphase domains (APD).

Journal ArticleDOI
TL;DR: A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed and should greatly facilitate the study of per oxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.

Journal ArticleDOI
TL;DR: Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose, indicating that a true collagenase had been isolated.
Abstract: Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose. Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield. Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated. The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind. The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee [Holmquist, B., & Vallee, B. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6216] that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue. In this case, the hydroxamic acid probably coordinates to the active-site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate. The IC50 for N-(benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes. The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases.

Journal ArticleDOI
TL;DR: A sugar oxidizing enzyme which produces H2O2 during glucose starvation in the white-rot fungus Phanerochaete chrysosporium has been purified from mycelial extracts and somewhat characterized, with possible importance of glucose-2-oxidase in lignin degradation.
Abstract: A sugar oxidizing enzyme which produces H2O2 during glucose starvation in the white-rot fungus Phanerochaete chrysosporium has been purified from mycelial extracts and somewhat characterized. Enzyme purity was confirmed by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis, both techniques revealing a homogeneous protein. The enzyme is active over a broad pH range with maximum activity at pH 7.5. Of several sugars tested, glucose was the preferred substrate although δ-d-gluconolactone and d-xylose were also oxidized at significant rates (at 60% and 37%, respectively, of the rate observed with glucose). Km-values for glucose and xylose are 1.03 and 20 mM respectively and the glucose oxidation product was idenitified as d-arabino-2-hexosulose. The possible importance of glucose-2-oxidase in lignin degradation is discussed.

Book ChapterDOI
TL;DR: This method permits the preparation of large amounts of stable, efficient homogeneous and well-defined substrate that is suitable for measuring the enzyme activity in plasma fractions.
Abstract: Publisher Summary Lecithin–cholesterol acyltransferase (LCAT) is synthesized by the liver and secreted into the bloodstream, where it is believed to act principally on the surface of high-density lipoproteins (HDL). The first attempts to isolate LCAT yielded only partially purified preparations even though a variety of chromatographic techniques were used, including hydroxylapatite adsorption, salt precipitation, ultracentrifugation, and affinity chromatography. Lecithin–cholesterol acyltransferase has been one of the most difficult enzymes to characterize from the physical, chemical, and enzymological points of view. The painstakingly slow early development of an efficient purification procedure and the instability of the enzyme precluded rapid progress. For the assay of LCAT activity, a stable, efficient, and uniform substrate that can be added to the enzyme source under conditions such that the concentration of substrate is not rate limiting is required. This method permits the preparation of large amounts of stable, efficient homogeneous and well-defined substrate that is suitable for measuring the enzyme activity in plasma fractions.

Journal ArticleDOI
TL;DR: In this article, the synthesis of a chiral substrate for possible organic conductors and superconductors is described, which is the state-of-the-art for chiral materials.
Abstract: Chiral metals are so far practically unknown. The synthesis of a chiral substrate for possible organic conductors and superconductors is described.

Journal ArticleDOI
TL;DR: Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form.

Journal ArticleDOI
TL;DR: It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions.

Journal ArticleDOI
01 Jun 1986-Diabetes
TL;DR: The results indicate that the human erythrocyte enzyme is very similar to the enzyme in other tissues and that these cells are a good source of material for purification of the human IDE.
Abstract: An insulin-degrading enzyme (IDE) was purified from the cytosol of human erythrocytes via the use of ammonium sulfate precipitation and chromatography on columns composed of DEAE-Sephadex, pentylagarose, hydroxylapatite, chromatofocusing resins, and Ultrogel AcA-34. The final preparation was purified greater than 50,000-fold and exhibited a single protein band of Mr = 110,000 on reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Cross-linking of 125I-labeled insulin to the enzyme preparation labeled a protein of the same molecular weight, indicating that this band was in fact the enzyme. Intact insulin, insulin B chain, and glucagon inhibited this cross-linking half-maximally at concentrations of 0.1, 1, and 1.5 microM, respectively. Under nondenaturing conditions, the enzyme had an Mr = 300,000, suggesting that the enzyme may exist under physiological conditions as a dimer or timer. The purified enzyme was inhibited by both sulfhydrylmodifying reagents and chelating agents, indicating that a free thiol and metal were both required for the activity of the enzyme. The purified enzyme was found to degrade physiological concentrations of intact insulin more rapidly than insulin B chain, although at high substrate concentrations (greater than 1 microM) the enzyme degraded B chain to a greater extent. Additional characteristics of the enzyme were a pl of 5.2 and a pH optimum of 7.0. These properties of the red blood cell (RBC) enzyme were very similar to those reported for IDEs from other tissues. Moreover, a polyclonal antiserum to the IDE from skeletal muscle was found to recognize the RBC enzyme.