SEARCH · PubMed Health

Results for “Photo-crosslinking”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed1979

An enzyme-polymer film prepared with the use of poly(vinyl alcohol) bearing photosensitive aromatic azido groups.

Photochemical reaction of poly(vinyl alcohol) bearing aromatic azido groups was applied for immobilization of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21.) in poly(vinyl alcohol) film. Photo-crosslinking and immobilization reactions proceeded by light irradiation for 25 min in air. The immobilized enzyme showed approx. 40% of its native enzyme activity with an apparent Michaelis constant of 3.9 mM. The Michaelis constant of the native enzyme was 2.3 mM. Some properties of the immobilized and native enzyme are compared.

Azides↗

PubMed1987

Novel Leu-Lys-specific peptidase (Leulysin) produced by gel-entrapped yeast cells.

Saccharomyces cerevisiae cells entrapped with a neutral hydrophilic photo-crosslinkable resin prepolymers specifically excreted into a cultured medium a new type of a peptidase, which cleaved Leu-Lys bond of alpha-mating factor. The enzyme was purified by membrane filtration followed by ion exchange chromatography and gel filtration chromatography. The purified enzyme showed a strict substrate specificity on internal Leu-Lys bond. Leu-Lys bond near terminus of a molecule and Leu-X and X-Lys bonds examined so far were not hydrolyzed by the enzyme.

Amino Acid Sequence↗

PubMed1995

Protein insertion into the endoplasmic reticulum of permeabilized cells.

We have established efficient translocation of newly synthesized proteins into the endoplasmic reticulum of permeabilized Mel Juso cells. By site-specific photo-crosslinking we show that translocating polypeptide chains contact the same components of permeabilized cells ER as in dog pancreas rough microsomes. This cellular assay system has the potential to overcome the limitations of isolated microsomes in investigating the molecular environment of a newly synthesized protein after they have left the ER translocation site.

Animals↗

PubMed1992

Topography of transcription factor complexes on the Saccharomyces cerevisiae 5 S RNA gene.

Locations of component proteins of yeast RNA polymerase III transcription factors (TFIII) A, C and B on a 5 S rRNA gene have been determined by site-specific DNA-protein photo-crosslinking. Comparison with a previously analyzed tRNA gene shows that similar nucleoprotein structures assemble on these two genes despite their differently located internal promoter elements. A principal signature of this homology is the placement of the 95 kDA subunit of TFIIIC, which associates with the box A promoter element of the tRNA gene. On the 5 S rRNA gene, the 95 kDa subunit occupies the same space in the absence of a box A sequence, and despite the presence of a box A-like sequence 30 base-pairs further downstream. A 90 kDa component that was not previously recognized as an integral part of TFIIIC has been specifically located at the 3' end of the 5 S rRNA gene.

Base Sequence↗

PubMed1986

Topoisomerase I interacts with transcribed regions in Drosophila cells.

The in vivo distribution of topoisomerase I on specific DNA sequences is determined at high resolution in Drosophila cells using a photo-crosslinking method. Topoisomerase I-DNA adducts are generated by irradiation of intact cells with UV light and then purified by immunoprecipitation with antibody to topoisomerase I. Analyses of the DNA sequences crosslinked to topoisomerase I by blot-hybridization with appropriate DNA probes indicate that topoisomerase I is concentrated on transcribed regions and not on nontranscribed flanking sequences. Like RNA polymerase II, topoisomerase I is recruited to heat-shock genes during the heat-shock response. However, topoisomerase I and RNA polymerase II can interact independently with the transcribed region because different ratios of topoisomerase I and RNA polymerase II are crosslinked to the highly transcribed hsp70 gene and the moderately transcribed copia genes. We hypothesize that topoisomerase I allows topological changes in DNA that are required for transcription.

Animals↗

PubMed1993

Design of bioluminescence-based fiber optic sensors for flow-injection analysis.

A fiber optic sensor based on enzyme-catalyzed light-emitting reactions has been developed and integrated in a flow-injection analysis (FIA) system. The firefly luciferase, specific for ATP, and the bacterial oxidoreductase/luciferase system, specific for NADH, have been immobilized on preactivated polyamide membranes. ATP and NADH analysis could be performed in the range from 0.1 pmol to 3 nmol and from 0.5 pmol to 1 nmol, respectively. By co-immobilizing these two bioluminescence systems on the same membrane, a multi-function biosensor has been designed allowing the alternate determination of ATP or NADH with the same sensitivity as that obtained with the two different mono-functional biosensors. A partly self-contained biosensor has been also developed for the flow injection analysis of NADH. For this purpose, FMN (one of the substrates of the bacterial bienzymatic system) has been embedded in a synthetic matrix. Different supports have been tested for the non-covalent immobilization of this substrate and its release in the immediate vicinity of the bound enzymes. Using a photo-crosslinked poly(vinyl alcohol) support, 40 reliable assays (CV = 4.5%) could be performed without changing or reloading the matrix.

Adenosine Triphosphate↗

PubMed1994

(1,3) beta-Glucan synthase activity of Neurospora crassa: identification of a substrate-binding protein.

(1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified 1300-fold to a specific activity of 14,000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions were extracted with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-beta-D-glucoside from Tergitol-extracted membrane preparations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)beta-glucan synthase enzyme complex. 5-Azido-[32P]UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)beta-glucan synthase.

Carrier Proteins↗

PubMed1991

Development of acetylcholine sensor using carbon fiber (amperometric determination).

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H2O2 which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several novel advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization and electrochemical measurements.

Acetylcholine↗

PubMedDate not supplied

Pitfalls in characterization of protein interactions using radioiodinated crosslinking reagents. Preparation and testing of a novel photochemical 125I-label transfer reagent.

Much attention has been focused on the study of protein interactions with radioiodinated photo-crosslinking reagents, and pitfalls in using this methodology are discussed. A new photochemical and cleavable heterobifunctional crosslinking reagent, succinimidyl N-14-(2-hydroxybenzoyl)-N-11-(4-azidobenzoyl)-9-oxo-8,11,14-triaza -4,5- dithiatetradecanoate (SHAD) was prepared, and its potential as a label transfer reagent was tested in model systems. SHAD was radioiodinated, and the labeled reagent (125I-SHAD) was converted to an amide (125I-HADM, as a mimicry of conjugation to protein 1) and photolyzed. When compared to the widely used SASD reagent (sulfosuccinimidyl 2-[[(4-azidosalicyl)-amino]ethyl]-1,3- dithiopropionate, Pierce), SHAD has a number of decisive advantages. The amide of 125I-SASD (125I-ASDM) was generated and photolyzed, and it was found that at least 50% of the radioactivity is released from 125I-ASDM after 3 min of irradiation, whereas only approximately 10% is liberated from 125I-HADM under similar conditions. Furthermore, 125I-HADM was photolyzed in the presence of excess amine (mimicry of crosslinking to protein 2), and the product was cleaved by reduction (mimicry of label transfer). The transformations in the course of photolysis were monitored by UV spectroscopy and TLC analysis, and a high degree of reagent cleavage upon reduction was demonstrated. 125I-SHAD was used to crosslink Lys78-plasminogen and fibrin. 125I-SHAD was conjugated to Lys78-plasminogen in the dark. Fibrinogen and thrombin were added, and Lys78-plasminogen was crosslinked to the fibrin clot by exposure to light.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗

PubMed1977

Photochemical attachment of cyclic AMP binding protein(s) to the nuclear genome.

The nature of the cyclic AMP-receptor-nucleus interactions was examined by a novel combination of two photoreactions. A photosensitive derivative of cyclic AMP, N6-butyryl cyclic AMP, was covalently attached to its cytoplasmic receptod by photo-affinity labelling and this receptor complex was photo-crosslinked by the DNA in the rat liver nuclei. The photolytic reactions seemed to be specific since stable links were formed only when substantial noncovalent binding occured.

Affinity Labels↗

PubMed1989

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site.

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.

Biological Transport↗

PubMed1984

Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

3-(4'-Benzoylphenyl)propionyl[3H] Phe-tRNA bound to the peptidyl site of the ribosome is photo-crosslinked exclusively to 23S RNA on irradiation at 320 nm. The site of reaction has been identified both by hybridization and primer-extension experiments as uridine-2584 and uridine-2585, located within the central loop of domain V according to the secondary structure model of 23S RNA. The fact that the covalently crosslinked tRNA retains its ability to form a peptide bond, together with the proximity of this site to the position of several mutations leading to chloramphenicol or erythromycin resistance strongly argue that this region of the 23S-like rRNAs is an integral component of the peptidyl transferase site. On the basis of these results, and from comparative analysis of the 16 available large subunit rRNA sequences, we propose a model for the functional organization of the peptidyl transferase site involving interaction of domains II and V of 23S rRNA.

Acyltransferases↗

PubMed1984

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA.

Wybutine (Ywye), situated next to the 3'-side of the anticodon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA, 5'-[32P]-labelled pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be crosslinked to the U at the 5'-position of the corresponding UUU-codon.

Base Sequence↗

PubMed1980

DNA strand specificity in promoter recognition by RNA polymerase.

DNA strand and enzyme subunit specificities involved in the interaction between E. coli RNA polymerase and T7 DNA were studied by photo-crosslinking techniques. In non-specific enzyme-DNA complexes, subunits, sigma, beta, and beta' were crosslinked to both strands of the DNA. Under conditions leading to specific enzyme-promoter complexes, however, only sigma and beta subunits were crosslinked. The sigma subunit was crosslinked preferentially to the non-sense strand at promoter sites. No such strand specificity was observed for the beta subunit. These results provide insight into the molecular mechanism of promoter recognition and indicate that the interaction between RNA polymerase and DNA template is different at promoters and at non-specific sites.

Binding Sites↗

PubMed1980

Photo-induced crosslinking between phenylalanine transfer RNA and messenger RNA on the Escherichia coli ribosome.

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at > 300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)-cellulose columns. The experiments suggest that the photo-crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome.

Escherichia coli↗

PubMed1993

Movement of the guide sequence during RNA catalysis by a group I ribozyme.

Ribozymes derived from the self-splicing pre-ribosomal RNA of Tetrahymena act as sequence-specific endonucleases. The reaction involves binding an RNA or DNA substrate by base pairing to the internal guide sequence (IGS) to form helix P1. Site-specific photo-crosslinking localized the 5' end of the IGS in helix P1 to the vicinity of conserved bases between helices P4 and P5, supporting a major feature of the Michel-Westhof three-dimensional structure model. The crosslinked ribozyme retained catalytic activity. When not base-paired, the IGS was still specifically crosslinked, but the major site was 37 A distant from the reactive site in the experimentally supported three-dimensional model. The data indicate that a substantial induced-fit conformational change accompanies P1 formation, and they provide a physical basis for understanding the transport of oligonucleotides to the catalytic core of the ribozyme. The ability of RNA to orchestrate large-scale conformational changes may help explain why the ribosome and the spliceosome are RNA-based machines.

Animals↗

PubMed1992

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.

Actins↗

PubMed1995

Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I.

DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.

Bacteriophage T7↗