Showing papers on "Substrate (chemistry) published in 1996"

Pulsed radio frequency plasma polymerization of allyl Alcohol: Controlled deposition of surface hydroxyl groups

Abstract: The utility of employing a variable duty cycle pulsed plasma polymerization technique to control film chemistry during plasma depositions is examined using allyl alcohol as monomer gas. Large scale progressive variations in film composition are observed with sequential changes in the plasma duty cycles employed, all other plasma variables being held constant. In particular, the −OH functionality of the monomer is increasingly retained in the plasma generated thin films as the radio frequency duty cycle is lowered. Fourier transform infrared and X-ray photoelectron spectroscopic analyses of the films obtained reveal that excellent film chemistry control is achieved during plasma polymerization of this monomer. The surface density controllability of functional groups, coupled with a gradient layering technique described herein to improve film adhesion to substrate surfaces, provides ideal opportunities for molecular tailoring of surfaces via subsequent derivatization reactions.

Humus and Enzyme Activity

Abstract: Publisher Summary A level of extracellular enzyme activity exists in soil. Enzymes secreted by living cells during normal cell activity, leaked from extant cells, or released from lysed cells, are short-lived unless they are adsorbed by soil colloids or inglobated by humic molecules. This level of extracellular enzyme activity indicates the biological capacity of the soil for the enzymatic conversion of the substrate, which is independent of the extant microbial activity; it also has an important role in the ecology of microorganisms. The activity of enzymes associated with mineral or organic colloids is one of the various activities contributing to the overall activity of the enzyme in soil. The extraction of humus–enzyme complexes from soil in good yields is obtained with solutions employed in extracting humic substances. The intrinsically high electron density of inorganic colloids masks the enzyme reaction product, while the humus material nonspecifically adsorbs the trapping agent becoming electron-dense. Using the electron probe microanalysis overcomes these problems. The chapter analyzes that if extracellular enzymes are observed in the humified organic matter, the comparison of data from extraction–purification experiments with observations by the electron probe microanalyses may prove useful.

Sensors for measuring analyte concentrations and methods of making same

Abstract: A sensor for measuring a concentration of an analyte in a solution. The sensor comprises a substrate layer, an electrode layer and an immobilized enzyme layer. The sensor further includes an enzyme/polymer layer and/or a hydrophobic layer. The enzyme/polymer layer includes an enzyme disposed within a polymer matrix. The hydrophobic layer is formed from a material that is more hydrophobic than the immobilized enzyme layer material. In certain embodiments, the sensor includes the enzyme/polymer layer and the hydrophobic layer disposed between the enzyme/polymer layer and the immobilized enzyme layer such that the enzyme/polymer layer is disposed along the hydrophobic layer within an area above the immobilized enzyme layer.

Chemical vapor deposition of metal sulfide films from metal thiocarboxylate complexes with monodentate or multidentate ligands

Abstract: In a method of depositing a metal sulfide film on a substrate (7), a solution containing at least one metal compound precursor comprising at least one thiocarboxylate ligand SECR, wherein E is selected from the group consisting of O and S and wherein R is selected from the group consisting of alkyl, aryl, substituted alkyl, substituted aryl, halogenated alkyl, and halogenated aryl is prepared. The substrate in a substrate chamber (4) is heated to a reaction temperature by a heating means (5). The solution is evaporated to form vapors of the metal compound precursor by the use of an aerosol generator (1). The vapors and the substrate heated to the reaction temperature are contacted. The reaction temperature is sufficient to decompose the metal compound precursor to form a metal sulfide film of at least one metal on the substrate.

Dewetting at the liquid-liquid interface.

Abstract: The dewetting behavior of a liquid film from a liquid substrate has been studied as a function of the substrate viscosity, using two highly viscous polymers as a model system. The dewetting velocity exhibits a minimum as a function of substrate viscosity. This behavior results from the competition of (1) the mass transport in the substrate, and (2) the retarded deformation of the liquid-liquid interface. Atomic force microscopy is used to image both the liquid-air interfaces and the buried liquid-liquid interface. The shape of the latter changes significantly with increasing substrate viscosity.

Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes.

Abstract: Human mitochondrial aldehyde dehydrogenase (ALDH-2) has a Km for acetaldehyde that is 900-fold lower than that for the cytosolic isozyme, ALDH-1. An increase in aliphatic aldehyde chain length decreases the ALDH-2 Km by up to 10-fold but decreases that of ALDH-1 by 5 orders of magnitude. As a consequence, the Km of ALDH-1 for decanal is 8 times lower than that of ALDH-2, i.e. 2.9 +/- 0.4 and 22 +/- 3 nM, respectively. Determination of these low Km values required kinetic analysis of the simultaneous enzymatic conversion of two aldehyde substrates, an approach also applied to aromatic and fused polycyclic aldehydes. For most of these substrates, maximum velocities are 5-100 times lower than those for acetaldehyde. Addition of one of these tight-binding, slow-turnover substrates to a reaction mixture containing ALDH, NAD+, and a "reference" aldehyde substrate (e.g. acetaldehyde) blocks the principal (reference) enzymatic reaction temporarily and reversibly. Once the first substrate is converted to product, the enzyme can act on the reference substrate. In terms of apparent affinity and blocking capacity, naphthalene and phenanthrene aldehydes were the most potent effectors. Other aromatic and fused polycyclic and heterocyclic aldehydes, as well as derivatives of coumarin, quinoline, indole, and pyridine, are tight-binding, slow-turnover substrates for ALDH-2 and relatively weak inhibitors of ALDH-1. The hydrophobicity of substituents of benzaldehydes, and particularly of naphthaldehydes, correlates with their binding constants toward ALDH-2. Vitamin A1 aldehydes are specific natural substrates for ALDH-1; at pH 7.5, for all-trans- and 13-cis-retinal, Km = 1.1 and 0.37 micromolar, respectively, and kcat/Km is 50-100 times higher than that for acetaldehyde. At the same time, the retinals are inhibitors of ALDH-2, all-trans-retinal being a particularly potent inhibitor (competitive Ki = 43 nM, noncompetitive Ki = 316 nM). These properties suggest that all-trans-retinal is a possible regulatory compound for ALDH-2 in vivo. The data in general point to specialized roles for both major human liver ALDH isozymes in the oxidation of bulky/hydrophobic natural compounds, with Km values in the low nanomolar range.

Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1.

Abstract: In the Finnerty pathway for n-alkane, oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, P. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires CU2+ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C12 to C20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.

Purification and Properties of Flavin- and Molybdenum-Containing Aldehyde Oxidase from Coleoptiles of Maize

Abstract: Aldehyde oxidase (AO; EC 1231) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L) seedlings The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300–600 nm) To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants

Kinetic characterization of the chymotryptic activity of the 20s proteasome

Abstract: In this paper, we report kinetic studies for the chymotryptic activity of the 20S proteasome. Major observations include the following: (1) Reaction progress curves that are recorded at concentrations of Suc-Leu-Leu-Val-Tyr-AMC greater than about 40 microM are biphasic and characterized by initial velocities that decay by a first-order process to final, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 40 microM, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate constant for the approach to steady-state has a complex dependence on substrate concentration and decreases sigmoidally as substrate concentration increases. These results indicate that the 20S proteasome is a hysteretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mechanistic features: (1) the 20S proteasome has two cooperative active sites for Suc-Leu-Leu-Val-Tyr-AMC and (2) there are two interconvertible conformers of active 20S proteasome. To probe this mechanism in greater detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC by the peptide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear dependence of reciprocal steady-state velocity on inhibitor concentration (i.e., parabolic inhibition) as well as a nonlinear dependence of the apparent inhibitor dissociation constant on substrate concentration. Both of these observations are explained by binding of inhibitor at multiple sites on the enzyme. Taken together, the results of this study indicate that the 20S proteasome is a conformationally flexible protein that can adjust to the binding of ligands and that has multiple and cooperative active sites. These results support a view of the proteasome's substrate specificity in which (1) substrates are recognized and hydrolyzed by more than one active site; (2) each active site can bind substrates that possess a variety of P1 residues; and (3) the P1 residue plays a relatively minor role as a specificity determinant. Finally, we interpret the results of this study to suggest that, in vivo, the 20S proteasome requires conformational plasticity for its interactions with regulatory complexes and, after it has combined with appropriate regulatory complexes, to catalyze hydrolysis of proteins.

Sensor and production method of and measurement method using the same

Abstract: There is provided a sensor for the measurement of a content of a material in liquid which material is oxidized with an oxidase enzyme in which sensor a reagent layer is formed on an electrode system composed of a measuring electrode and a counter electrode both of which are formed on an insulating substrate, the reagent layer is composed of a hydrophilic polymer layer comprising a hydrophilic polymer and a reactive layer comprising the oxidase enzyme and an electron carrier, and the reagent layer further comprises a phosphate.

A Novel Optical Complex between an Organic Substrate and a Polyoxometalate. Crystal and Molecular Structure of α-H4SiW12O40·4HMPA·2H2O (HMPA = Hexamethylphosphoramide)

Abstract: A solvated complex of α-H4SiW12O40·4HMPA·2H2O composed the heteropolytungstate α-H4SiW12O40 and the organic substrate hexamethylphosphoramide (HMPA) has been synthesised, purified, and characterized. The electronic spectra (λ = 220−500 nm) as well as the 1H NMR spectra for the title compound dissolved in CD3CN establish that this complex dissociates into free SiW12O404- and HMPA moieties in solution unless the organic substrate HMPA is present in very high concentrations. The solid reflectance electronic spectra and IR spectra indicate that there is interaction between the α-H4SiW12O40 and the organic substrate. The complex has no photosensitivity under irradiation of sunlight, but under the near-UV light result in a charge transfer by oxidation of the HMPA and the reduction of the polyoxometalate. Light yellow polyhedrons of the title compound crystallize from the aqueous solvent of acetonitrile and aqueous solution as the formula of α-H4SiW12O40·4HMPA·2H2O in the monoclinic, space group P21. The unit ce...

Microcrystalline-cellulose hydrolysis with concentrated sulphuric acid

Abstract: The effects of temperature (25-40°C), H,SO, concentration (31-70% (w/v)) and the acid/substrate relationship (1-5 cm3 of H,SO, per g-' of cellulose) on the solubilization rate of microcrystalline cellulose and on the glucose production rate have been analysed. The solubilization process was by determining reducing groups present in solution. For acid/substrate relationships of more than 1 cm3 g-' and H,SO, concentrations of greater than 62% (w/v), the acid promoted the total solubilization of the cellulose in the form of chains with a low degree of polymerization within 4 h. The solubilization demonstrated zero-order kinetics in which the specific rate and time of total solubilization are a function of the variables in operation. Glucose was produced according to a mechanism of two consecutive first-order pseudo-homogeneous reactions. The values of the kinetic constants k, and k, have been correlated with temperature, the H,SO, concentration and the acid/substrate relationship.

A perspective on biological catalysis.

Abstract: We have analysed enzyme catalysis through a re-examination of the reaction coordinate. The ground state of the enzyme-substrate complex is shown to be related to the transition state through the mean force acting along the reaction path; as such, catalytic strategies cannot be resolved into ground state destabilization versus transition state stabilization. We compare the role of active-site residues in the chemical step with the analogous role played by solvent molecules in the environment of the noncatalysed reaction. We conclude that enzyme catalysis is significantly enhanced by the ability of the enzyme to preorganize the reaction environment. This complementation of the enzyme to the substrate's transition state geometry acts to eliminate the slow components of solvent reorganization required for reactions in aqueous solution. Dramatically strong binding of the transition state geometry is not required.

A Carboxylate Oxygen of the Substrate Bridges the Magnesium Ions at the Active Site of Enolase: Structure of the Yeast Enzyme Complexed with the Equilibrium Mixture of 2-Phosphoglycerate and Phosphoenolpyruvate at 1.8 Å Resolution†,‡

Abstract: The equilibrium mixture of yeast enolase with substrate, 2-phospho-d-glycerate (2-PGA), and product, phosphoenolpyruvate (P-enolpyruvate), has been crystallized from solutions of poly(ethylene glycol) (PEG) at pH 8.0. Crystals belong to the space group C2 and have unit cell dimensions a = 121.9 A, b = 73.2 A, c = 93.9 A, and β = 93.3°. The crystals have one dimer per asymmetric unit. Crystals of the equilibrium mixture and of the enolase complex of phosphonoacetohydroxamate (PhAH) are isomorphous, and the structure of the former complex was solved from the coordinates of enolase−(Mg2+)2−PhAH [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333−9342]. The current crystallographic R-factor is 17.7% for all recorded data (92% complete) to 1.8 A resolution. The electron density map is unambiguous with respect to the positions and liganding of both magnesium ions and with respect to the stereochemistry of substrate/product binding. Both magnesium ions are complexed to functi...

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

Abstract: It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to fluoro-cis-aconitate, followed by addition of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05-A resolution, release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10(6)-fold excess of isocitrate, and previously published Mossbauer experiments. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative alpha-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds < 2.7 A in length involving HTn, H2O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.

Chemical amplification positive type resist material

Abstract: PROBLEM TO BE SOLVED: To provide a chemical amplification positive type resist material for electronic beam or soft x-ray exposure which is a chemical amplification positive type resist material containing one or more kinds of an alkali-insoluble or an slightly alkali-soluble resins introducing an acid-labile group to a part of hydrogen atoms of carboxyl groups or phenol hydroxyl groups of a base polymer soluble in an alkaline solution containing a carboxyl group or a phenol hydroxyl group and which is characterized in that the above resin contains at least two kinds of acid-labile groups, one being an acetal group or a ketal group and the other being a tertiary hydrocarbon group or a substituent containing a tertiary hydrocarbon group. SOLUTION: The resist material of the present invention is excellent in stability when left in vacuum after exposure with electronic beams and is low in trailing on a Cr substrate and is excellent in sensitivity, degree of resolution and plasma etching resistance. Thus, the resist material of the present invention is especially suitable for a material for forming a minute pattern in machining of a mask substrate by these characteristics.

Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar‐beet pulp

Abstract: An inducible esterase has been isolated from a liquid culture of Aspergillus niger grown on sugar-beet pulp. The enzyme was active on methyl esters of cinnamic acids, caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase. The enzyme was not active on methyl sinapinate, a good substrate for ferulic acid esterase III, which was purified previously from A. niger [Faulds and Williamson (1994) Microbiology 140, 779-787]. With methyl caffeate as substrate the enzyme had temperature and pH optima of 50 degrees C and 6.0 respectively, and a specific activity of 96.9 units per mg of protein. The purified protein (native molecular mass 145 000 Da) gave a single heavily stained band on SDS/PAGE, suggesting the protein was a dimer, and seemed to be heavily glycosylated. Isoelectric focusing gave a single band corresponding to a pl of 4.80. The pure enzyme was free of other carbohydrase activities. The activity of the pure enzyme was inhibited by more than 99% after treatment with the serine-specific protease inhibitor aminoethylbenzenesulphonylfluoride (1 mM) for 12 h. The enzyme was capable of releasing ferulic acid from sugar beet pulp.