Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Knoevenagel condensation of 2-carbethoxycyclohexanone and malononitrile: Synthesis of 4-cyano-3-ethoxy-1-hydroxy-5,6,7,8-tetrahydroisoquinoline, an isomer of the abnormal product

The structure of the abnormal product 1a formed in the Knoevenagel condensation of 2-carbethoxycyclohexanone and malononitrile has been further confirmed. Oxidation of the tetrahydroisoquinoline 3b using Na2Cr2O-AcOH-H2SO4 gave the keto isoquinoline 3d and the isoquinoline-1-carboxylic acid 5a. The acid chloride of 5a was condensed with diethyl ethoxymagnesiomalonate to afford after decarbethoxylation the methyl ketone 5d which on Baeyer-Villiger oxidation gave a mixture of the acetate 1g and the title compound 1b. The unambiguous synthesis of 1b confirms the structure assigned earlier to the title compound also formed during the partial hydrolysis of the diethoxy compound 1c. Condensation of 2-acetylcyclohexane-1,3-dione with malononitrile gave the quinoline derivative 4c which on ethylation yielded the ketoquinoline 4d. The present studies have confirmed that the quinoline compound 4a is also formed in the condensation of 2-acetylcyclohexanone and cyanoacetamide.

Bromination of Intermolecular Chelate Linkage Isomers of 3-Oximino-4,9-Dimethyl (or 4,7,9-Trimethyl)-5,8-Diazadodeca-4,8-Diene-2,11-Dionato Nickel(II)

The reaction of the title complexes (FIG. 1) with N-bromosuccinimide or bromine in chloroform yields isomeric bromo complexes on substitution of the γ-CH carbon proton by bromine. The brominated products have been characterised by ir, pmr, electronic absorption spectra, conductivity and magnetic susceptibility measurements. The linkage isomerisation of the brominated products in chloroform has been shown to depend on the diamine residue.

Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv.

Two methods were employed to measure the rate of ribonucleic acid (RNA) chain growth in vivo in Mycobacterium tuberculosis H37Rv cultures growing in Sauton medium at 37 degrees C, with a generation time of 10 h. In the first, the bacteria were allowed to assimilate [3H]uracil or [3H]guanine into their RNA for short time periods. The RNA was then extracted and hydrolyzed with alkali, and the radioactivity in the resulting nucleotides and nucleosides was measured. The data obtained by this method allowed the calculation of the individual nucleotide step times during the growth of RNA chains, from which the average rate of RNA chain elongation was estimated to be about 4 nucleotides per s. The second method employed the antibiotic rifampin, which specifically inhibits the initiation of RNA synthesis without interfering with the elongation and completion of nascent RNA chains. Usint this method, the transcription time of the 16S, 23S, and 5S ribosomal RNA genes was estimated to be 7.6 min, which corresponds to a ribosomal RNA chain growth rate of 10 nucleotides per s.

Studies in terpenoids. Part XXXI. Synthesis of pyrocurzerenone, a furosesquiterpenoid from Curcuma zedoaria

7-Acetonyloxy-5-methyl--tetralone (Vc) was cyclodehydrated to 7,8-dihydro-1,5-dimethylnaphtho[2,1-b]furan-9(6H)-one (VIa), the structure of which was established by an independent synthesis from methyl 4-(4-acetonyloxy-2-methylphenyl)butyrate (IXd). Similarly, 7-acetonyloxy-2,5-dimethyl--tetralone (Vf), synthesized via 4-(5-isopropyl-4-methoxy-2-methylphenyl)-2-methylbutyric acid (XIIb) and 7-methoxy-2,5-dimethyl--tetralone (Vd), was cyclodehydrated to 7,8-dihydro-1,5,8-trimethylnaphtho[2,1-b]furan-9(6H)-one (VIb), which on reduction and dehydration furnished pyrocurzerenone (6,7-dihydro-1,5,8-trimethylnaphtho[2,1-b]furan)(I). The deisopropylation and cyclodehydration of (XIIb) to (Vd) were effected in one step by treatment with polyphosphoric acid.

A One-Step Method for the Growth of Ga2O3-Nanorod-Based White-Light-Emitting Phosphors

A one-step synthesis of Ga2O3 nanorods by heating molten gallium in ambient air at high temperatures is presented. The high-temperature synthesis creates oxygen vacancies and incorporates nitrogen from the environment. The oxygen vacancy in Ga2O3 is responsible for the emission in the blue-green region, while nitrogen in Ga2O3 is responsible for red emission.

Response of a turbulent boundary layer to a step change in surface heat flux

Measurements of both the velocity and the temperature field have been made in the thermal layer that grows inside a turbulent boundary layer which is subjected to a small step change in surface heat flux. Upstream of the step, the wall heat flux is zero and the velocity boundary layer is nearly self-preserving. The thermal-layer measurements are discussed in the context of a self-preserving analysis for the temperature disturbance which grows underneath a thick external turbulent boundary layer. A logarithmic mean temperature profile is established downstream of the step but the budget for the mean-square temperature fluctuations shows that, in the inner region of the thermal layer, the production and dissipation of temperature fluctuations are not quite equal at the furthest downstream measurement station. The measurements for both the mean and the fluctuating temperature field indicate that the relaxation distance for the thermal layer is quite large, of the order of 1000θ0, where θ0 is the momentum thickness of the boundary layer at the step. Statistics of the thermal-layer interface and conditionally sampled measurements with respect to this interface are presented. Measurements of the temperature intermittency factor indicate that the interface is normally distributed with respect to its mean position. Near the step, the passive heat contaminant acts as an effective marker of the organized turbulence structure that has been observed in the wall region of a boundary layer. Accordingly, conditional averages of Reynolds stresses and heat fluxes measured in the heated part of the flow are considerably larger than the conventional averages when the temperature intermittency factor is small.

Furazans and furazan oxides. 7. Interconversions of anthranils, benzofurazan oxides, and indazoles

7-Nitroanthranil (1, R = R = H) and 4-formylbenzofurazan oxide (2, R = R' = H) equilibrate on heating. The latter condenses with primary amines and the resulting imines rearrange to 7-nitroindazoles (8). The corresponding 6-methoxy and 6-chloro derivatives of 1 behave similarly. Neither 5- nor 6-nitroanthranil forms an indazole on heating with aniline or other primary amines.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

A Remarkably Simple One-Step Procedure for the Preparation of alpha-Bromo-alpha,beta-Unsaturated Carbonyl Compounds

An easy and convenient one-step procedure for the conversion of alpha,beta-unsaturated carbonyl compounds into their corresponding bromo-enones using NBS-Et3N center dot 3HBr in the presence of potassium carbonate in dichloromethane at 0 degrees C to room temperature under very mild conditions in high yields and significantly shorter times, is reported.

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

An FETI-preconditioned conjuerate gradient method for large-scale stochastic finite element problems

In the spectral stochastic finite element method for analyzing an uncertain system. the uncertainty is represented by a set of random variables, and a quantity of Interest such as the system response is considered as a function of these random variables Consequently, the underlying Galerkin projection yields a block system of deterministic equations where the blocks are sparse but coupled. The solution of this algebraic system of equations becomes rapidly challenging when the size of the physical system and/or the level of uncertainty is increased This paper addresses this challenge by presenting a preconditioned conjugate gradient method for such block systems where the preconditioning step is based on the dual-primal finite element tearing and interconnecting method equipped with a Krylov subspace reusage technique for accelerating the iterative solution of systems with multiple and repeated right-hand sides. Preliminary performance results on a Linux Cluster suggest that the proposed Solution method is numerically scalable and demonstrate its potential for making the uncertainty quantification Of realistic systems tractable.

Convenient Synthesis of Ferrocene Conjugates Mediated by Benzyltriethylammonium Tetrathiomolybdate in a Multi-Step Tandem Process

The synthesis of a wide range of ferrocene-derived sulfur-linked mono- and disubstituted Michael adducts and conjugates mediated by benzyltriethylammonium tetrathiomolybdate (1) in a tandem process is reported. New route to access acryloylferrocene (4) and 1,1'-diacryloylferrocene (5) is discussed. Conjugation of amino acids to ferrocene is established via their N and C termini and also via side chains employing conjugate addition as key step to furnish mono-and divalent conjugates. This methodology has also been extended to access several ferrocene-carbohydrate conjugates. The electrochemical behavior of some selected ferrocene conjugates was studied by cyclic voltammetry.

Ethyl 4-(3-hydroxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate

In the molecular structure of the title compound, C21H25NO4, the dihydropyridine ring adopts a flattened boat conformation while the cyclohexenone ring is in an envelope conformation. In the crystal structure, molecules are linked into a two-dimensional network parallel to (10 (1) over bar) by N-H center dot center dot center dot O and O-H center dot center dot center dot O hydrogen bonds. The network is generated by R-4(4)(30) and R-4(4)(34) graph-set motifs.