Mammalian brain plasma membranes: Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na+-K+) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.

The nonplanar peptide unit III. Quantum chemical calculations for related compounds and experimental X-ray diffraction data

The possible nonplanar distortions of the amide group in formamide, acetamide, N-methylacetamide, and N-ethylacetamide have been examined using CNDO/2 and INDO methods. The predictions from these methods are compared with the results obtained from X-ray and neutron diffraction studies on crystals of small open peptides, cyclic peptides, and amides. It is shown that the INDO results are in good agreement with observations, and that the dihedral angles N and defining the nonplanarity of the amide unit are correlated approximately by the relation N = -2, while C is small and uncorrelated with . The present study indicates that the nonplanar distortions at the nitrogen atom of the peptide unit may have to be taken into consideration, in addition to the variation in the dihedral angles (,), in working out polypeptide and protein structures.

Chemical modification studies on Abrus agglutinin Involvement of tryptophan residues in sugar binding

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydratebinding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2- hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

A single point mutation disrupts the capsid assembly in Sesbania Mosaic Virus resulting in a stable isolated dimer

Protein-protein interactions play a Crucial role in Virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried Out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable Soluble dimers of the capsid Protein. The X-ray crystal structure of one of the isolated dimer mutants - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. Detailed analysis of the dimeric mutant protein structure revealed that a number of Structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated chiller was ``more relaxed'' than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the Virus, has different conformations in the isolated dimer and the intact Virus Suggesting its flexible nature and the conformational changes that accompany assembly. The isolated choler mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal Structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.

A model for doped-oxide-source diffusion with a chemical reaction at the silicon-silicon dioxide interface

A mathematical model for doped-oxide-source diffusion is proposed. In this model the concept of segregation of impurity at the silicon-silicon dioxide is used and also a constant of “rate limitation” is introduced through a chemical reaction at the interface.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group.

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.

Modeling of Freezing Phenomena Induced by Chemical Reactions

Spatial variations in the concentration of a reactive solute in solution are often encountered in a catalyst particle, and this leads to variation in the freezing point of the solution. Depending on the operating temperature, this can result in freezing of the solvent oil a portion of catalyst, rendering that part of the active area ineffective Freezing call occur by formation of a sharp front or it mush that separates the solid and fluid phases. In this paper, we model the extent of reduction in the active area due to freezing. Assuming that the freezing point decreases linearly with solute concentration, conditions for freezing to occur have been derived. At steady state, the ineffective fraction of catalyst pellet is found to be the same irrespective of the mode of freezing. Progress of freezing is determined by both the heat of reaction and the latent heat of fusion Unlike in freezing of alloys where the latter plays a dominant role, the exothermicity of the reaction has a significant effect on freezing in the presence of chemical reactions. A dimensionless group analogous to the Stefan number could be defined to capture the combined effect of both of these.

LbL Fabricated Poly(Styrene Sulfonate)/TiO2 Multilayer Thin Films for Environmental Applications

Fabrication of multilayer ultrathin composite films composed of nanosized titanium dioxide particles (P25, Degussa) and polyelectrolytes (PELs), such as poly(allyl amine hydrochloride) (PAH) and poly(styrene sulfonate sodium salt) (PSS), on glass substrates using the layer-by-layer (LbL) assembly technique and its potentia application for the photodegradation of rhodamine B under ultraviolet (UV) irradiation has been reported. The polyelectrolytes and TiO2 were deposited on glass substrates at pH 2.5 and the growth of the multilayers was studied using UV/vis speccrophotometer. Thicknes measurements of the films showed a linear increase in film thickness with increase in number of bilayers. The surface microstructure of the thin films was characterized by field emission scanning electron microscope. The ability of the catalysts immobilized by this technique was compared with TiO2 films prepared by drop casting and spin coating methods. Comparison has been made in terms of film stability and photodegradation of rhodamine B. Process variables such as the effect of surface area of the multilayers, umber of bilayers, and initial dye concentration on photodegradation of rhodamine B were studied. Degradation efficiency increased with increase in number of catalysts (total surface area) and bilayers. Kinetics analysis indicated that the photodegradation rates follow first order kinetics. Under maximum loading of TiO2, with five catalyst slides having 20 bilayers of polyelectrolyte/TiO2 on each, 100 mL of 10 mg/L dye solution could be degraded completely in 4 h. The same slides could be reused with the same efficiency for several cycles. This study demonstrates that nanoparticles can be used in wastewater treatment using a simple immobilization technique. This makes the process an attractive option for scale up.

Surfactant-Mediated Synthesis of Functional Metal Oxide Nanostructures Via Microwave Irradiation-Assisted Chemical Synthesis

In the present work we report a rapid microwave irradiation-assisted chemical synthesis technique for the growth of nanoparticles, nanorods, and nanotubes of a variety of metal oxides in the presence of an appropriate surfactant (cationic, anionic, non ionic and polymeric), without the use of any templates. The method is simple, inexpensive, and helps one to prepare nanostructures in quick time, measured in seconds and minutes. This method has been applied successfully to synthesize nanostructures of a variety of binary and ternary metal oxides such as ZnO, CdO, Fe2O3, CuO, Ga2O3, Gd2O3, ZnFe2O4, etc. There is an observed variation in the morphology of the nanostructures with changes in different process parameters, such as microwave power, irradiation time, identity of solvent, type of surfactant, and its concentration.

H N.M.R. studies of protected alpha-aminoisobutyric acid containing peptides. Chemical shift nonequivalence of benzyloxycarbonyl methylene protons

The benzylic methylene protons in a large number of benzyloxycarbonyl alpha-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 leads to 1 hydrogen bonded beta-turn is not possible, the benzylic-CH2-protons appear as a singlet in CDCl3 and have a very small chemical shift difference in (CD3)2SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro beta-turn is retained in large peptide-fragments of alamethicin.

High lithium storage in micrometre sized mesoporous spherical self-assembly of anatase titania nanospheres and carbon

Composite of anatase titania (TiO2) nanospheres and carbon grown and self-assembled into micron-sized mesoporous spheres via a solvothermal synthesis route are discussed here in the context of rechargeable lithium-ion battery. The morphology and carbon content and hence the electrochemical performance are observed to be significantly influenced by the synthesis parameters. Synthesis conditions resulting in a mesoporous arrangement of an optimized amount carbon and TiO2 exhibited the best lithium battery performance. The first discharge cycle capacity of carbon-titania mesoporous spheres (solvothermal reaction at 150 degrees C at 6 h, calcination at 500 degrees C under air, BET surface area 80 m(2)g(-1)) was 334 mAhg(-1) (approximately 1 Li) at current rate of 0.066 Ag-1. High storage capacity and good cyclability is attributed to the nanostructuring of TiO2 (mesoporosity) as well as due to formation of a percolation network of carbon around the TiO2 nanoparticles. The micron-sized mesoporous spheres of carbon-titania composite nanoparticles also show good rate cyclability in the range (0.066-6.67) Ag-1.

Chemical modifications of natural triterpenes—glycyrrhetinic and boswellic acids: evaluation of their biological activity

Synthetic analogues of naturally occurring triterpenoids; glycyrrhetinic acid, arjunolic acid, and boswellic acids, by modification of A-ring with a cyano- and enone-functionality, have been reported. A novel method of synthesis of α-cyanoenones from isoxazoles is reported. Bioassays using primary mouse macrophages and tumor cell lines indicate potent anti-inflammatory and cytotoxic activities associated with cyano-enones of boswellic acid and glycyrrhetinic acid.

Direct probe of end-segment distribution in tethered polymer chains

End-tethered chains made of an adsorbed diblock copolymer of polystyrene (PS)-polyisoprene (PI) bearing an end-segment including a Ge atom are built by the Langmuir-Schaeffer technique. They are studied both in the dry state and in a good solvent for the PI chain using grazing incidence X-ray standing waves. The analysis of the signal provides a direct measurement of the end-segment distribution which is found to be singular and mostly localized to a plane in the dry case. In the good solvent case, end-segments are found to span the entire assembly and compare very well with results obtained by Kreer et al.