Orientation of transmembrane polypeptides as revealed by antibody quenching of fluorescence.
AuthorsBar-Noy S, Darmon A, Ginsburg H, Cabantchik ZI
JournalBiochim Biophys Acta
PubMed ID6391545
'We describe here a new method, based on fluorescent techniques, for the determination of the orientation of membrane protein molecules present in vesicles. The method consists of: (a) attachment of a fluorescein derivative to sugar residues of glycoproteins and glycolipids in the cell membrane, and (b) the use of anti-fluorescein ... More
Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence.
AuthorsDarmon A, Bar-Noy S, Ginsburg H, Cabantchik ZI
JournalBiochim Biophys Acta
PubMed ID3893545
'The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by ... More
Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein.
AuthorsKabisch UC, Gräntzdörffer A, Schierhorn A, Rücknagel KP, Andreesen JR, Pich A
JournalJ Biol Chem
PubMed ID10085076
'Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N ... More
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore. 1. The 3' terminus of 16S RNA.
AuthorsFriedrich K, Woolley P
JournalEur J Biochem
PubMed ID2833391
'We have investigated the use of the pH-sensitive fluorescein label as a probe for electrostatic potential in macromolecules. The practicality of this technique is demonstrated by its application to the 16S RNA molecule. The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and ... More
Redistribution of plasma-membrane surface molecules during formation of the Leishmania amazonensis-containing parasitophorous vacuole.
AuthorsHenriques C, de Souza W
JournalParasitol Res
PubMed ID10726992
'Leishmania amazonensis presents two developmental stages that gain access to the host macrophage through phagocytosis. The protozoan resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which can fuse with the endocytic system. For evaluation of the parasite/host-cell interaction process and of PV biogenesis, the two parasite forms or host-cell ... More
The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances.
AuthorsOdom OW, Dabbs ER, Dionne C, Müller M, Hardesty B
JournalEur J Biochem
PubMed ID6378636
'The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3'' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic ... More
Human erythrocytes adhering to schistosomula of Schistosoma mansoni lyse and fail to transfer membrane components to the parasite.
AuthorsCaulfield JP, Cianci CM
JournalJ Cell Biol
PubMed ID4008525
'We studied the adherence of human erythrocytes to larvae of the intravascular parasite Schistosoma mansoni by transmission microscopy, freeze fracture, and fluorescence techniques. In addition, we used the adherent cells to investigate the problem of host antigen acquisition. Schistosomula were cultured for from 24 to 48 h after transformation in ... More
Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.
AuthorsCorbett JD, Cho MR, Golan DE
JournalBiophys J
PubMed ID8130343
'We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with ... More
Monopalmitoylphosphatidylcholine incorporation into human erythrocyte ghost membranes causes protein and lipid immobilization and cholesterol depletion.
AuthorsGolan DE, Furlong ST, Brown CS, Caulfield JP
JournalBiochemistry
PubMed ID3401442
'The effects of lysophosphatidylcholine (lysoPC) on human erythrocyte (RBC) ghost morphology, transmembrane protein and lipid lateral mobilities, and membrane lipid composition were studied in order to elucidate mechanisms by which lysoPC immobilizes ghost membrane components [Golan, D. E., Brown, C. S., Cianci, C. M. L., Furlong, S. T., & Caulfield, ... More
Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.
AuthorsGoto T, Wong KS, Brunette DM
JournalJ Histochem Cytochem
PubMed ID10544222
'Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured ... More
Molecular basis of RNA recognition by the embryonic polarity determinant MEX-5.
AuthorsPagano JM, Farley BM, McCoig LM, Ryder SP
JournalJ Biol Chem
PubMed ID17264081
'Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an ... More
Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes.
'Escherichia coli 16S RNA from 30S ribosomal subunits was isolated, oxidized at the 3'' end, and labeled with the thiosemicarbazide derivatives of fluorescein or eosin. Labeled 16S RNA was reconstituted into 30S subunits. They were almost fully active compared to 30S subunits reconstituted from unlabeled 16S RNA by using a ... More
Protein carbonyl formation in blood plasma by cephalosporins.
AuthorsJung Y, Chay K, Song D, Yang S, Lee M, Ahn B
JournalArch Biochem Biophys
PubMed ID9308904
'Cephalosporin antibiotics caused the formation of carbonyl groups in the plasma proteins both in vivo and in vitro. After the administration of either moxalactam (3 g/day) or cefotaxime (2 g/day) to patients for 7 days, the carbonyl contents in the plasma proteins increased markedly as determined by the 2,4-dinitrophenylhydrazine (DNPH) ... More
Labeling of the glycoprotein subunit of (Na,K)ATPase with fluorescent probes.
AuthorsLee JA, Fortes PA
JournalBiochemistry
PubMed ID2983755
'Sodium plus potassium activated adenosinetriphosphatase [(Na,K)ATPase] is composed of a catalytic subunit (alpha) and a glycoprotein subunit (beta) of unknown function. A method has been developed to label the beta subunit of purified dog kidney (Na,K)ATPase with fluorescent probes. The method consists of oxidation of beta-subunit oligosaccharides, reaction of the ... More
Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes.
'The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3'' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3'' ends of ... More
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore. 2. Transfer RNA.
AuthorsFriedrich K, Woolley P, Steinhäuser KG
JournalEur J Biochem
PubMed ID3281833
'The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA. By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to ''map'' the electrostatic ... More
Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.
AuthorsBakin AV, Borisova OF, Shatsky IN, Bogdanov AA
JournalJ Mol Biol
PubMed ID1717698
'The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors ... More
The cercarial glycocalyx of Schistosoma mansoni.
AuthorsSamuelson JC, Caulfield JP
JournalJ Cell Biol
PubMed ID2985622
'Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx ... More
A simple and non-invasive visualization for assessment of carbonylated protein in the stratum corneum.
AuthorsFujita H, Hirao T, Takahashi M
JournalSkin Res Technol
PubMed ID17250537
'BACKGROUND/PURPOSE: Stratum corneum (SC) is the interface of body and environment and is continuously exposed to oxidative stress, resulting in oxidative modification of proteins. Consequent carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and subsequently detected with anti-DNP antibody. We developed a simpler, non-invasive method to ... More
A simple method for labelling the carbohydrate moieties of antibodies with fluorochromes.
AuthorsDuijndam WA, Wiegant J, Van Duijn P, Haaijman JJ
JournalJ Immunol Methods
PubMed ID2452205
A fluorescence-labeling method for sequencing small RNA on polyacrylamide gel.
AuthorsWu TP, Ruan KC, Liu WY
JournalNucleic Acids Res
PubMed ID8811106
A practical fluorescence-labeling method for sequencing small RNAs by the traditional 'direct read out' on polyacrylamide gel electrophoresis was established. The 3' terminus of RNA was oxidized into dialdehyde by sodium periodate and then labeled with fluorescein-5-thiosemicarbazide through the condensation reaction between carbazide and aldehyde. The fluorescence-labeled RNA was partially ... More
Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes.
AuthorsPagano JM, Clingman CC, Ryder SP,
JournalRNA
PubMed ID21098142
Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. ... More
Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.
AuthorsStöffler-Meilicke M, Stöffler G, Odom OW, Zinn A, Kramer G, Hardesty B
JournalProc Natl Acad Sci U S A
PubMed ID7029538
Periodate-oxidized 3' ends of 5S, 23S, and 16S rRNAs from Escherichia coli were allowed to react with fluorescein thiosemicarbazide, then labeled rRNAs were reconstituted into active ribosomal subunits. The fluorescein moiety on each of the rRNAs when reconstituted into ribosomal subunits was accessible to anti-fluorescein IgG as determined by fluorescence ... More
Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels.
AuthorsAhn B, Rhee SG, Stadtman ER
JournalAnal Biochem
PubMed ID2883911
Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably ... More
Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli.
AuthorsCzworkowski J, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID2029524
Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes. The interaction of these labeled RNAs with the 30S subunit of ... More
Fluorimetric distance determination by resonance energy transfer. Ribosome-bound transfer RNA.
AuthorsFriedrich K, Woolley P, Steinhäuser KG
JournalEur Biophys J
PubMed ID3396517
Using the technique of singlet-singlet (Förster-type) resonance energy transfer, we have determined five distances in the programmed ribosome, either with the P site or with both the A and the P sites occupied. Two of the distances are new and two agree with earlier measurements; the fifth showed disagreement in ... More
Distance measurement by energy transfer: the 3' end of 16-S RNA and proteins S4 and S17 of the ribosome of Escherichia coli.
Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyl and/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3' end of ... More
Ribosome binding by tRNAs with fluorescent labeled 3' termini.
AuthorsWells BD, Cantor CR
JournalNucleic Acids Res
PubMed ID6160468
Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X ... More
Interaction and thermal stability of fluorescent labeled derivatives of thrombin and antithrombin III.
AuthorsAtha DH, Brew SA, Ingham KC
JournalBiochim Biophys Acta
PubMed ID6696918
Derivatives of human thrombin and antithrombin III with fluorescent labels covalently attached to their carbohydrate moieties were prepared by reaction of periodate-oxidized proteins with amino derivatives of dansyl, fluorescein and pyrene. The labeled derivatives retained full biological activity, including their ability to form stable enzyme-inhibitor complexes, a reaction whose rate ... More
Localization of the elongation factor Tu binding site on Escherichia coli ribosomes.
AuthorsRychlik W, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID6338919
Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a ... More
A new strategy for site-specific protein modification: analysis of a Tat peptide-TAR RNA interaction.
AuthorsTamilarasu N, Zhang J, Hwang S, Rana TM
JournalBioconjug Chem
PubMed ID11312672
Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-alpha-Fmoc-3-acetyl-L-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino ... More
Escherichia coli L-aspartate-alpha-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry.
AuthorsRamjee MK, Genschel U, Abell C, Smith AG
JournalBiochem J
PubMed ID9169598
The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a ... More
The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction.
Authorsde-Souza W, de-Carvalho TU, de-Melo ET, Soares CP, Coimbra ES, Rosestolato CT, Ferreira SR, Vieira M
JournalBraz J Med Biol Res
PubMed ID9921284
In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein ... More
Redistribution of parasite and host cell membrane components during Toxoplasma gondii invasion.
AuthorsPacheco-Soares C, De Souza W
JournalCell Struct Funct
PubMed ID9706405
The initial association of tachyzoites of Toxoplasma gondii with a host cell induces an endocytic process which leads to the formation of a vacuole known as the parasitophorous vacuole (PV). We analyzed the parasite-host cell interaction process using either parasites or host cells whose membrane was previously labeled with probes ... More
Fluorescence studies of the location of proteins L7 and L12 on the Escherichia coli ribosome.
AuthorsLee CC, Wells BD, Fairclough RH, Cantor CR
JournalJ Biol Chem
PubMed ID7005217
The location of ribosomal protein L7/L12 has been examined by fluorescence spectroscopy. Protein L7 modified mainly at the COOH terminus by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonic acid, and yeast tRNAPhe-containing fluorescent dyes located specifically near the anticodon or at the 3'-end were used in these studies. Fluorescence-quenching measurements indicate that the COOH terminus of ... More
Substrate-specific selenoprotein B of glycine reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.
AuthorsWagner M, Sonntag D, Grimm R, Pich A, Eckerskorn C, Söhling B, Andreesen JR
JournalEur J Biochem
PubMed ID10091582
The substrate-specific selenoprotein B of glycine reductase (PBglycine) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (alpha), 25 (beta) and 47 kDa (gamma), probably in an alpha 2 beta 2 gamma 2 composition. PBglycine purified from cells grown ... More
Localization of noncovalently bound ethidium in free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer.
AuthorsFerguson BQ, Yang DC
JournalBiochemistry
PubMed ID3639742
Ethidium binds tRNAfMet with 17-fold enhancement in the emission intensity at 600 nm. Fluorescence titration of tRNAfMet with ethidium indicates a single high-affinity site in tRNAfMet with a dissociation constant of 5 microM. Ethidium is apparently rigidly bound to tRNAfMet and effectively shielded from solvent. tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) with ... More
Comparison of ribosomal entry and acceptor transfer ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAPhe to the 3' end of 16S ribonucleic acid.
AuthorsRobbins D, Hardesty B
JournalBiochemistry
PubMed ID6197085
Distances were measured by nonradiative energy transfer from fluorescent probes specifically located on one of three points of yeast or Escherichia coli Phe-tRNAPhe enzymatically bound to the entry site or to the acceptor site of E. coli 70S ribosomes to energy-accepting probes on the 3' end of the 16S ribonucleic ... More
Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.
AuthorsAinger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, Carson JH
JournalJ Cell Biol
PubMed ID7691830
We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case ... More
Store operated Ca2+ influx by selective depletion of ryanodine sensitive Ca2+ pools in primary human skeletal muscle cells.
AuthorsWeigl L, Zidar A, Gscheidlinger R, Karel A, Hohenegger M
JournalNaunyn Schmiedebergs Arch Pharmacol
PubMed ID12690427
The contraction and relaxation of skeletal muscle is driven by release of Ca2+ from sarcoplasmic reticulum through the ryanodine receptor type 1 and extruding the ion from the cytosol by Ca2+ ATPases. Efficient refilling of the empty Ca2+ stores is essential for repetitive cycles of muscle contraction and relaxation, but ... More
Domain interactions in the gelatinase A.TIMP-2.MT1-MMP activation complex. The ectodomain of the 44-kDa form of membrane type-1 matrix metalloproteinase does not modulate gelatinase A activation.
AuthorsOverall CM, Tam E, McQuibban GA, Morrison C, Wallon UM, Bigg HF, King AE, Roberts CR
JournalJ Biol Chem
PubMed ID10991943
On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from ... More
Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components.
AuthorsGolan DE, Brown CS, Cianci CM, Furlong ST, Caulfield JP
JournalJ Cell Biol
PubMed ID3745271
Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane ... More