A solid phase assay for the protease of human immunodeficiency virus

A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.     

Acetate C-C bond formation and decomposition in the anaerobic world: the structure of a central enzyme and its key active-site metal cluster

The structure of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS), a central enzyme in the anaerobic metabolism of acetyl-coenzyme A (acetyl-CoA), has been solved to a resolution of 2.2A. The active-site metal cluster responsible for catalyzing acetyl C-C bond synthesis and cleavage, designated the A center, was identified as an Fe(4)S(4) iron sulfur cluster with one of its cysteine thiolates acting as a bridge to an adjacent binuclear metal site. Nickel was found at one position in the binuclear site and the other metal was indicated to be copper - a surprising result, implying a previously unrecognized role for copper. Details of the A center provided new insight into the unusual organometallic mechanism of acetyl C-C bond formation and cleavage, with substantial conformational changes indicated for binding of the large methylcorrinoid protein substrate, and a unique intramolecular channel acting to contain carbon monoxide within the protein and transfer it to the site needed for acetyl-CoA synthesis.     

Nanoscale features of fibronectin fibrillogenesis depend on protein-substrate interaction and cytoskeleton structure

Cell-reorganized fibronectin layers on polymer films providing a gradation of the binding strength between protein and substrate were analyzed by combined fluorescence and scanning force microscopy. The nanoscale fibronectin patterns exhibited paired parallel fibrils with characteristic spacings of 156, 233, 304, and 373 nm. These spacings depend on the interaction of fibronectin with the substrate: at enhanced fibronectin-substrate anchorage the cells form larger stress fibers, which are assembled by alpha-actinin cross-linked pairs of actin filaments subunits at the focal adhesions. A ubiquitous repeating unit of approximately 71 nm was found within these characteristic distances. We conclude that the dimensions of the actin stress fibers reflect the binding strength of fibronectin to the polymer substrate and act--in turn--as a template for the reorganization of fibronectin into surface-bound nanofibrils with characteristic spacings. This explanation was confirmed by data showing the alpha-actinin/fibronectin colocalization.     

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis

Atomic resolution (相似文献   

The role of hydrogen bonding in the enzymatic reaction catalyzed by HIV-1 protease

The hydrogen-bond network in various stages of the enzymatic reaction catalyzed by HIV-1 protease was studied through quantum-classical molecular dynamics simulations. The approximate valence bond method was applied to the active site atoms participating directly in the rearrangement of chemical bonds. The rest of the protein with explicit solvent was treated with a classical molecular mechanics model. Two possible mechanisms were studied, general-acid/general-base (GA/GB) with Asp 25 protonated at the inner oxygen, and a direct nucleophilic attack by Asp 25. Strong hydrogen bonds leading to spontaneous proton transfers were observed in both reaction paths. A single-well hydrogen bond was formed between the peptide nitrogen and outer oxygen of Asp 125. The proton was diffusely distributed with an average central position and transferred back and forth on a picosecond scale. In both mechanisms, this interaction helped change the peptide-bond hybridization, increased the partial charge on peptidyl carbon, and in the GA/GB mechanism, helped deprotonate the water molecule. The inner oxygens of the aspartic dyad formed a low-barrier, but asymmetric hydrogen bond; the proton was not positioned midway and made a slightly elongated covalent bond, transferring from one to the other aspartate. In the GA/GB mechanism both aspartates may help deprotonate the water molecule. We observed the breakage of the peptide bond and found that the protonation of the peptidyl amine group was essential for the peptide-bond cleavage. In studies of the direct nucleophilic mechanism, the peptide carbon of the substrate and oxygen of Asp 25 approached as close as 2.3 A.     

几丁聚糖在硅橡胶表面作涂层的实验研究

几丁聚糖具有良好的生物相容性和抗菌性,作为生物涂层已经引起了广泛的注意。研究了基体的不同表面处理方法、浸涂次数和几丁聚糖溶液浓度对涂层的表面形貌、结合强度等性能的影响,结果显示,几丁聚糖溶液浓度和硅橡胶表面粗糙度都存在一个最佳取值范围;增加浸涂次数可以改善涂层光洁度,但是对涂层附着力贡献不大;硅橡胶经紫外照射后可以改善几丁聚糖在其上的成膜性能。     

Asymmetric alkene reduction by yeast old yellow enzymes and by a novel Zymomonas mobilis reductase

The genes encoding yeast old yellow enzymes (OYE 1, 2, and 3) and NAD(P)H-dependent 2-cyclohexen-1-one reductase from Zymomonas mobilis (NCR) were expressed separately in Escherichia coli. All four recombinant strains reduced the carbon double bond in alpha,beta-unsaturated alkenals and alkenones, however rates and enantio-specificities differed. Which of the two possible enantiomers was predominantly formed, was not only dependent on the choice of enzyme but also on the substrate: In addition to a dependency on methylation in alpha- or beta-position, the data of this study illustrate that firstly the E- or Z-configuration (cis- or trans-) of the carbon double-bond and secondly the remainder of the substrate molecule play roles in determining enantio-specificity. Based on the currently accepted mechanism of flavin mediated anti-hydrogenation of the carbon double bond, the data in this study may be explained by a flipped orientation of some of the substrates in the active center of OYE.     

Extended substrate recognition in caspase-3 revealed by high resolution X-ray structure analysis

Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Angstroms) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side-chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Angstroms crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction.     

Kinetic resolution of organosilicon compounds by stereoselective dehydrogenation with horse liver alcohol dehydrogenase

Streoselective dehydrogenation of three isomers of trimethylsilypropanol was carried out with horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1.) and optically active organosilicon compounds were obtained in a water-organic solvent two-layer system with coenzyme regeneration. Furthermore, we examined the effects of the silicon atom on stereoselectivity of HLADH compared to the corresponding carbon compounds. Substitution of the silicon atom for the carbon atom was found to improve the stereoselectivity of HLADH. For example, the optical purity of the remaining 1-trimethylsilyl-2-propanol was higher than 99% enantiomeric excess (ee) at 50% conversion, whereas that of the carbon analogue was 84% ee. This phenomenon was probably ascribable to the bulkiness of the organosilicon compounds derived from their longer Si-C bond. Kinetic analysis in an aqueous monolayer system demonstrated that the specific properties of the silicon atom greatly affected the reactivity of these substrate compounds.Correspondence to: A. Tanaka     

Kinetics of the inhibition of thrombin by hirudin

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.     

Purification and characterization of proteinase yscJ, a new yeast peptidase.

A newly recognized peptidase, designated proteinase yscJ, was purified from the yeast Saccharomyces cerevisiae. The enzyme is of non-vacuolar origin and cleaves the Tyr-Lys bond of the synthetic peptide substrate Cbz-Tyr-Lys-Arg-NH-Ph (Cbz, benzyloxycarbonyl; NH-Ph, 4-nitroanilide) and the Glu-Lys bond of the substrate Boc-Glu-Lys-Lys-NH-Mec (Boc, butoxycarbonyl; Mec, 4-methylcoumarinyl) with high efficiency. Optimum pH for cleavage of Cbz-Tyr-Lys-Arg-NH-Ph is in the range 7.0-7.5. The purified enzyme has a molecular mass of approximately 58 kDa, as judged by gel filtration on a Superose 12 FPLC column. Mercury compounds and EDTA were found to be potent inhibitors of proteinase yscJ activity.     

Carbon post-microarrays for glucose sensors

A novel design and fabrication method of glucose sensors based on high aspect ratio carbon post-microarrays is reported in this paper. Apart from the fact that carbon has a wide electrochemical stability window, a major advantage of using carbon post-microarrays as working electrodes for an amperometric glucose sensor is the large reactive surface per unit footprint substrate area, improving sensitivity of the glucose sensor. The carbon post-microarrays were fabricated by carbon-microelectromechanical systems (C-MEMS) technology. Immobilization of enzyme onto the carbon post-electrodes was carried out through co-deposition of glucose oxidase (GOx) and electrochemically polymerized polypyrrole (PPy). Sensing performance of the glucose sensors with different post-heights and various post-densities was tested and compared. The carbon post-glucose sensors show a linear range from 0.5 mM to 20 mM and a response time of about 20 s, which are comparable to the simulation result. Sensitivity per unit footprint substrate area as large as 2.02 mA/(mM cm²) is achieved with the 140 μm high (aspect ratio around 5:1) carbon post-samples, which is two times the sensitivity per unit footprint substrate area of the flat carbon films. This result is consistent with the hypothesis that the number of reaction sites scales with the reactive surface area of the sensor. Numerical simulation based on enzymatic reaction and glucose diffusion kinetics gives the optimum geometric design rules for the carbon post-glucose sensor. Glucose sensors with even higher sensitivity can be achieved utilizing higher carbon post-microarrays when technology evolution will permit it.     

Process modelling and simulation of a transketolase mediated reaction: Analysis of alternative modes of operation

A previously proposed model of a transketolase catalysed carbon–carbon bond formation reaction condensing β-hydroxypyruvate and glycolaldehyde to synthesise l-erythrulose has been extended to describe various modes of operation as an alternative to a batch process. The alternative continuous and fed-batch operations, with each substrate being fed separately and together have been analysed using the extended model. The analysis was carried out simulating the product concentration after a given time under defined process conditions. Comparison of product concentration and yield on catalyst as two process metrics were used to identify promising cases for further process development.     

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance

During the process of terpene biosynthesis, C–C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the ‘biosynthetic history’ of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ¹³C value for FDP, based on δ¹³C values and relative amounts of the products, was identical with its measured δ¹³C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon–carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without ‘touching’ the plant.     

Effects of Type I Collagen Degradation on the Durability of Three Adhesive Systems in the Early Phase of Dentin Bonding

ObjectiveThis study was designed to evaluate the effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding.MethodsBonded dentin specimens were prepared using three different types of adhesive systems. Micro-tensile bond strength and degradation of collagen were tested before, and after 1 month or 4 months of aging in artificial saliva. The relationship between micro-tensile bond strength and collagen degradation was analyzed by calculating their Pearson’s correlation coefficient.ResultsAging induced time-dependent reduction in micro-tensile bond strengths for all the tested adhesive systems, although such reduction for the single-step self-etching adhesive G-Bond (GB) was not statistically significant. The bond strength of the two-step self-etching primer adhesive system Clearfil SE Bond (SEB) was similar to that of the two-step etch-and-rinse self-priming adhesive system Single Bond 2 (SB), and they were both significantly reduced after one or four months of aging. A negative correlation was found between the degree of collagen degradation and magnitude of micro-tensile bond strength (r = - 0.65, p = 0.003). The Pearson’s correlation coefficient was 0.426, indicating that 42.6% of the aging-induced reduction in bond strength can be explained by the degradation of collagen.ConclusionsIn the early phase of dentin bonding, there was a negative correlation between the degree of collagen degradation and the magnitude of micro-tensile bond strength. The reduction of bond strength was accompanied by the degradation of collagen. These results provide evidence for the causative relationship between the degradation of collagen and the deterioration of dentin-adhesive interface.     

Proteinase yscD. Purification and characterization of a new yeast peptidase

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.     

Substrate specificity of the serine proteinase from Bacillus subtilis, strain 72

A comparative study of the hydrolysis of various p-nitroanilide substrates (Z-A2-A1-pNA, Z-A3-A2-A1-pNA, and Z-A4-A3-A2-A1-pNA, where A1-An are various amino acid residues, Z is the benzoyloxycarbonylic group and pNA is the p-nitroanilide group), catalyzed by serine proteinase from Bacillus subtilis strain 72, was carried out. It was found that depending on the substrate structure, the hydrolysis may involve both the peptide-p-nitroaniline and the amino acid-amino acid bonds. A kinetic analysis of substrate hydrolysis occurring simultaneously at these two bonds was carried out. The physico-chemical meaning of the kinetic parameters of the given scheme was determined. The quantitative estimation of the enzyme specificity with respect to both hydrolyzing bonds can be found by using the parameters calculated during the analysis of the kinetic curve of p-nitroaniline production. It was found that according to their specificity the amino acid residues at position A1 can be arranged in the following order: L-Leu greater than P-Phe greater than L-Ile greater than L-Ala. The beta-branched amino acid residues, L-Val and L-Ile, do not bind to subsite S1. If these residues occupy position A1, the substrate splitting occurs exclusively between residues A1 and A2. The tetrapeptide N-protected p-nitroanilide substrates are also hydrolyzed at this bond. Partial hydrolysis of the amino acid-amino acid bond between residues A1 and A2 occurs in two cases: i) when residue A1 is loosely bound to subsite S1 and/or, ii) when residue A2 is firmly bound to subsite S1.     

Applications of chromic acid-celite columns to lipid analysis. Location of double bond position in submicro- and microgram amounts of methyl octadecenoates.

A procedure for locating the double bond position in a series of methyl octadecenoates is detailed. Submicrogram or microgram amounts of substrate dissolved in CS₂ are brought in contact with a very small column of chromic acid on Celite, and the oxidation products (car?ylic acids) are eluted, converted to methyl esters, and resolved by gas-liquid chromatography. Beside the acids resulting from scission of the double bond, acids containing one less carbon atom arise from oxidation of the allylic carbons on both sides of the double bond so that pairs of peaks appear on the chromatogram. All positions from Δ3 to Δ17 were located successfully. The Δ2 position failed to oxidize.     

Polymer composite fluorescent hydrogel film based on nitrogen‐doped carbon dots and their application in the detection of Hg2+ ions

A simple microwave‐assisted solvothermal method was used to prepare fluorescent nitrogen‐doped carbon dots (N‐CDs) with high fluorescence quantum yield (79.63%) using citric acid and N‐(2‐hydroxyethyl)ethylenediamine as starting materials. The PVAm‐g‐N‐CDs grafted products were synthesized by amide bond formation between the carboxylic groups of N‐CDs and amine groups of polyvinylamine (PVAm). Fluorescent hydrogel films (PVAm‐g‐N‐CDs/PAM) were synthesized by interpenetration polymer network polymerization of PVAm‐g‐N‐CDs and acrylamide (AM). When used for ion detection, we found that the fluorescence of the hydrogel films was clearly quenched by addition of Hg²⁺. Repeatability tests on using the hydrogel films for Hg²⁺ detection showed that they could be applied at least three times. The PVAm‐g‐N‐CDs/PAM could serve as an effective fluorescent sensing platform for sensitive detection of Hg²⁺ ions with a detection limit of 0.089 μmol/L. This work may offer a new approach for developing recoverable and sensitive N‐CDs‐based sensors for biological and environmental applications.     

Langmuir-Blodgett films of a novel cellulose derivative with dihydrophytyl group: the ability to anchor beta-carotene molecules

A novel cellulose derivative, 6-O-dihydrophytylcellulose (DHPC), was first synthesized via a ring-opening polymerization and allowed to self-assemble onto an air-water interface. Langmuir-Blodgett (LB) films were characterized with atomic force microscope (AFM), UV-vis spectroscopy, and Fourier transform infrared spectroscopy. The surface pressure-area (pi-A) isotherms for DHPC and beta-carotene (betaC) mixture indicated strong interaction between these compounds to pack well. Thus, DHPC has the ability to anchor betaC in the monolayer. It was proved that a betaC-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films by UV spectroscopic analysis. The transmission and reflection-absorption IR spectra (RAS) indicated that the dihydrophytyl chains had almost trans-zigzag conformation and were oriented nearly perpendicular to the substrate. AFM section analysis revealed the thickness per layer to be 2.32 nm. Consequently, DHPC was found to be an appropriate matrix to fabricate the mixed LB films containing betaC.