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Biomedical subjects

G M Whitesides

Publications and source records attributed to G M Whitesides.

At least 91 records · Page 5Linked to original sources
PubMed1993

Neuraminidase-resistant hemagglutination inhibitors: acrylamide copolymers containing a C-glycoside of N-acetylneuraminic acid.

Copolymers consisting of a polyacrylamide backbone with side chains terminated in C-glycosidic analogs of N-acetylneuraminic acid were synthesized by free radical copolymerization of alpha-2-C-[3-[[2-(N-acryloylamino)ethyl]thio]propyl]-N- acetylneuraminic acid (5) with acrylamide. Unlike natural and synthetic polyvalent materials that contain N-acetylneuraminic acid in O-glycosidic form, these C-glycosidic copolymers resist neuraminidase-catalyzed cleavage of the neuraminic acid residue from the copolymer backbone. Examination of these C-glycosidic copolymers in a hemagglutination inhibition assay indicated that they are as effective in vitro as polyvalent O-glycosidic copolymers in inhibiting agglutination of erythrocytes by influenza virus. The minimum value of the inhibition constant, calculated on the basis of the concentration of Neu5Ac groups in solution, is Ki(HAI) approximately 10(-7) M for both copolymers. The inhibitory potency of the C-glycoside-based copolymers becomes more significant at lower concentrations of Neu5Ac moieties in solution than does the inhibitory potency of the O-glycoside-based copolymer.

Acrylamides↗
PubMed1993

Use of affinity capillary electrophoresis to determine kinetic and equilibrium constants for binding of arylsulfonamides to bovine carbonic anhydrase.

Affinity capillary electrophoresis (ACE) provides a new approach to studying protein-ligand interactions. The basis for ACE is the change in the electrophoretic mobility of the protein when it forms a complex with its ligand. This binding interaction can be quantified directly for charged ligands or indirectly for neutral ligands in competition with a previously characterized charged ligand. Determination of kinetic and equilibrium constants using ACE relies only on the changes in the migration time and shape (but not the area) of the peak due to protein. Simulation of the protein mobility under conditions of ACE suggests that the experimentally obtained electropherograms can be explained in terms of few variables: on and off rates (and thus, binding constant), concentration of the ligand(s), and relative mobilities of the protein and its complex(es).

Animals↗
PubMed1993

A convenient procedure for transfer blotting of coomassie blue stained proteins from PAGE gels to transparencies.

Proteins stained with Coomassie Brilliant Blue I were transferred effectively by blotting from polyacrylamide gel electrophoresis (PAGE) gels to transparencies of the type used in plain-paper copiers. The details of the original electropherogram were retained on transfer and did not fade over a period of three years. Both the protein and the associated dye transfer; however, protein does not transfer in the absence of dye. Protein patterns present in several types of gels--sodium dodecyl sulfate (SDS)-PAGE, non-denaturing PAGE, isoelectric focusing PAGE and SDS-agarose--all transferred successfully after staining with dye 1. Proteins visualized with other organic dyes such as Fast Green FCF 2, Uniblue A 3 and Procion Blue MX-R 4 also transferred, but proteins stained with Stains-all 5 or silver did not. This transfer provides a simple, economical way to preserve data from slab gel electrophoresis and a convenient method to display data using an overhead projector. The blotted transparencies are also excellent substrates for use with gel scanning densitometers.

Coloring Agents↗
PubMed1991

Molecular self-assembly and nanochemistry: a chemical strategy for the synthesis of nanostructures.

Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds. Molecular self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecular surfaces in biological aggregates is a central concern in structural biochemistry. Self-assembly is also emerging as a new strategy in chemical synthesis, with the potential of generating nonbiological structures with dimensions of 1 to 10(2) nanometers (with molecular weights of 10(4) to 10(10) daltons). Structures in the upper part of this range of sizes are presently inaccessible through chemical synthesis, and the ability to prepare them would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.

Biotechnology↗
PubMed1991

Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces.

Self-assembled monolayers (SAMs) of omega-functionalized long-chain alkanethiolates on gold films are excellent model systems with which to study the interactions of proteins with organic surfaces. Monolayers containing mixtures of hydrophobic (methyl-terminated) and hydrophilic [hydroxyl-, maltose-, and hexa(ethylene glycol)-terminated] alkanethiols can be tailored to select specific degrees of adsorption: the amount of protein adsorbed varies monotonically with the composition of the monolayer. The hexa(ethylene glycol)-terminated SAMs are the most effective in resisting protein adsorption. The ability to create interfaces with similar structures and well-defined compositions should make it possible to test hypotheses concerning protein adsorption.

Adsorption↗
PubMed1991

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.

Amino Acid Sequence↗
PubMed1990

Enzyme-catalyzed organic synthesis of sucrose and trehalose with in situ regeneration of UDP-glucose.

This paper describes cell-free enzymatic syntheses of sucrose and trehalose using partially-purified preparations of sucrose and trehalose synthetase. The coupling of the regeneration of uridine-5'-diphosphoglucose (UDP-Glc) with synthesis of the disaccharide offers a practical route to millimol quantities of these carbohydrates. The syntheses used pyruvate kinase, UDP-Glc pyrophosphorylase, and inorganic pyrophosphatase, and the regenerated UDP-Glc was cycled approximately 10 times.

Catalysis↗
PubMed1990

Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase.

This paper (1) describes the enzymatic synthesis of a mixture of adenosine, guanosine, cytidine, and uridine triphosphates (ATP, GTP, CTP, and UTP) from ribonucleic acid (RNA). RNA was hydrolyzed by nuclease P1 to a mixture of 5'-nucleoside monophosphates. This mixture was converted to the nucleoside triphosphates using a mixture of nucleoside monophosphate kinases and acetate kinase, with acetyl phosphate as the ultimate phosphoryl donor. The nucleoside monophosphokinases were extracted from brewer's yeast in a four-step procedure. The specific activity of the yeast enzyme preparation after gel permeation chromatography was sufficiently high that the yeast kinases could be immobilized in volumes that were practical for laboratory scale syntheses. Conversions from NMP to NTP in a mixture containing 0.34 mol of total nucleoside phosphates were: ATP, 90%; GTP, 90%; CTP, 60%; and UTP, 40%.

Acetate Kinase↗
PubMed1989

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study.

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.

Carbohydrate Sequence↗
PubMed1987

Regeneration of nicotinamide cofactors for use in organic synthesis.

The high cost of nicotinamide cofactors requires that they be regenerated in situ when used in preparative enzymatic synthesis. Numerous strategies have been tested for in situ regeneration of reduced and oxidized cofactors. Regeneration of reduced cofactors is relatively straightforward; regeneration of oxidized cofactors is more difficult. This review summarizes methods for preparation of the cofactors, factors influencing their stability and lifetime in solution, methods for their in situ regeneration, and process considerations relevant to their use in synthesis.

Kinetics↗
PubMed1987

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation.

Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0 degrees C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4 by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein synthesis could be improved by approximately 50% by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.

Animals↗