Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.
AuthorsHsi KL, O'Neill SA, Dupont DR, Yuan PM
JournalAnal Biochem
PubMed ID9527845
'A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated ... More
Simple adaptations to extend the range of flow cytometry five orders of magnitude for the DNA analysis of uni-and multicellular systems.
AuthorsHecht RM, Schomer DF, Oró JA, Bartel AH, Hungerford EV
JournalJ Histochem Cytochem
PubMed ID7019314
'Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The method employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier ... More
Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.
AuthorsScherson T, Kreis TE, Schlessinger J, Littauer UZ, Borisy GG, Geiger B
JournalJ Cell Biol
PubMed ID6547721
'Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated ... More
Fluorescence of fluorescein attached to myosin SH1 distinguishes the rigor state from the actin-myosin-nucleotide state.
AuthorsAndo T
JournalBiochemistry
PubMed ID6546523
'It has been found that the fluorescence intensity of 5-(iodoacetamido)fluorescein (5-IAF) attached to the SH1 of myosin subfragment 1 (S-1) increases 3-fold on formation of the rigor complex. On adding Mg2+-ADP, light scattering indicates no dissociation, but the fluorescence increment disappears. Thus, this fluorescence signal can distinguish the rigor state ... More
Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites.
AuthorsBoskovic DS, Krishnaswamy S
JournalJ Biol Chem
PubMed ID10984491
'The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to ... More
Dynamics of bacteriophage T4 presynaptic filament assembly from extrinsic fluorescence measurements of Gp32-single-stranded DNA interactions.
AuthorsLiu J, Qian N, Morrical SW
JournalJ Biol Chem
PubMed ID16829679
'In the bacteriophage T4 homologous recombination system, presynaptic filament assembly on single-stranded (ssDNA) DNA requires UvsX recombinase, UvsY mediator, and Gp32 ssDNA-binding proteins. Gp32 exerts both positive and negative effects on filament assembly: positive by denaturing ssDNA secondary structure, and negative by competing with UvsX for ssDNA binding sites. UvsY ... More
Probing the sulfhydryl groups of nuclear matrix proteins with 6-iodoacetamidofluorescein.
AuthorsMartelli AM, Billi AM, De Marchis C, Manzoli L, Cocco L
JournalCell Biol Int Rep
PubMed ID2357731
'Rat liver nuclear matrices were reacted with the fluorescent dye 6-iodoacetamidofluorescein and the matrix proteins were then separated by one and two-dimensional polyacrylamide gel electrophoresis. Upon transillumination with U.V. light it was possible to see that several proteins had reacted with the dy, thus indicating the presence of free -SH ... More
Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence.
AuthorsFadden P, Haystead TA
JournalAnal Biochem
PubMed ID7539987
'Reaction conditions were defined for the selective quantitative derivatization and fluorophore labeling of phosphoserine residues on peptides and proteins. Phosphoserine was derivatized with 1,2-ethanedithiol using a modification of the reaction conditions defined by R. C. Clark and J. Dijkstra (1967) Int. J. Biochem. 11, 577-585 and H. E. Meyer, E. ... More
Cell-mediated lympholytic responses against autologous cells modified with haptenic sulfhydryl reagents. III. Different regions of hapten-membrane protein conjugates influence CTL specificity and Ir gene control of hapten-self CTL responses.
Interaction between pyridoxal kinase and pyridoxine-5-P oxidase, two enzymes involved in the metabolism of vitamin B6.
AuthorsKwok F, Churchich JE
JournalJ Biol Chem
PubMed ID6243300
Optical chemical imaging of tobacco mosaic virus in solution at 60-nm resolution.
AuthorsKeller TH, Rayment T, Klenerman D
JournalBiophys J
PubMed ID9545066
Scanning near-field optical microscopy can provide images with a resolution less than the wavelength of light, and therefore ought in principle to be of great value in studies of biological structures. In this work we show how for the first time images have been obtained of tobacco mosaic virus particles ... More
Exposure of actin thiols by the removal of tightly held calcium ions.
AuthorsKonno K, Morales MF
JournalProc Natl Acad Sci U S A
PubMed ID3865205
The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves ... More
An easy and efficient fluorescent method for detecting aldehydes and its application in biotransformation.
AuthorsXing Y, Wang S, Mao X, Zhao X, Wei D,
JournalJ Fluoresc
PubMed ID20953822
Water-soluble aldehydes (acetaldehyde, propionaldehyde) and non-water-soluble aldehydes (butyraldehyde and phenylacetaldehyde) were easily detected by an efficient fluorescent method with 5-aminofluorescein as probe. Under optimal detection conditions, 5-aminofluorescein could selectively respond to aldehydes with high sensitivity in comparison with other carbonyl compounds like ketones and acids. Thus, the proposed method was ... More
Assay of membrane complement receptors (CR1 and CR2) with C3b- and C3d-coated fluorescent microspheres.
AuthorsLambris JD, Ross GD
JournalJ Immunol
PubMed ID7033372
A sensitive and specific fluorescence assay for membrane complement (C) receptors (CR1 and CR2) was developed with purified C3b and C3d fragments coupled to fluorescent microspheres (0.9 mu diameter). C3-microspheres (C3-ms) bound to cells with low numbers of receptors that were undetectable by other assay techniques. Inhibition studies with anti-CR1 ... More
Uptake of latex particles by macrophages: characterization using flow cytometry.
AuthorsParod RJ, Brain JD
JournalAm J Physiol
PubMed ID6614156
Flow cytometry can be used to characterize the uptake of particles by small samples of pulmonary macrophages both quickly and accurately. We found that there was a linear relationship between the number of fluorescent latex particles and the fluorescent intensity associated with each cell up to 47 particles/cell. Macrophages lavaged ... More
9-Anthroylnitrile binding to serine-181 in myosin subfragment 1 as revealed by FRET spectroscopy and molecular modeling.
AuthorsSzarka K, Bódis E, Visegrády B, Nyitrai M, Kilár F, Somogyi B
JournalBiochemistry
PubMed ID11732899
It has been shown that one of the 12 serine residues within the 23 kDa segment of myosin subfragment 1 can be covalently modified with a fluorescent probe 9-anthroylnitrile (ANN) [Hiratsuka, T. (1989) J. Biol. Chem. 264 (30), 18188-18194]. To identify the exact binding site of the probe, the distances ... More
Changes in nucleosome structure and histone H3 accessibility. Iodoacetamidofluorescein labelling after treatment with phosphatidylserine vesicles.
AuthorsCocco L, Martelli AM, Billi AM, Matteucci A, Vitale M, Neri LM, Manzoli FA
JournalExp Cell Res
PubMed ID2427349
The accessibility of the sulfhydryl-specific dye 6-iodoacetamidofluorescein (IAF) to H3 histone has been studied in isolated rat liver nuclei and mononucleosomal core particles after treatment with phosphatidylserine (PS) multilamellar vesicles (MLV). In isolated nuclei, despite the enhancement of total RNA synthesis and the massive chromatin decondensation produced by liposomes, the ... More
Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy.
AuthorsChen CS, Chen WN, Zhou M, Arttamangkul S, Haugland RP
JournalJ Biochem Biophys Methods
PubMed ID10737220
Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A ... More
The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II).
AuthorsGriep MA, McHenry CS
JournalBiochemistry
PubMed ID3048397
The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding ... More
An iodoacetamide spin-label selectively labels a cysteine side chain in an occluded site on the sarcoplasmic reticulum Ca(2+)-ATPase.
AuthorsWawrzynow A, Collins JH, Coan C
JournalBiochemistry
PubMed ID8399229
Sarcoplasmic reticulum vesicles were labeled with [14C]iodoacetamide spin-label (ISL) under conditions where time courses of the reaction predicted that one amino acid residue would be preferentially labeled. Solubilized tryptic peptides were separated by high-performance liquid chromatography following extensive digestion, and amino acid sequences were determined for major and minor radio-labeled ... More
6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.
Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a ... More
Fluorescent dyes for lymphocyte migration and proliferation studies.
AuthorsParish CR
JournalImmunol Cell Biol
PubMed ID10571670
Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the ... More
Decoration of microtubules by fluorescently labeled microtubule-associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts.
AuthorsVandenbunder B, Borisy GG
JournalCell Motil Cytoskeleton
PubMed ID3802218
Microtubule-associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization ... More
The tumor suppressor protein Fhit. A novel interaction with tubulin.
FHIT (fragile histidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. ... More
Spatial orientation and dynamics of the U1A proteins in the U1A-UTR complex.
AuthorsClerte C, Hall KB
JournalBiochemistry
PubMed ID10852733
The human U1A protein contains three distinct domains: the N-terminal RBD1 (amino acids 1-101), the C-terminal RBD2 (amino acids 195-282), and the linker region (amino acids 102-194). The RBD1 domains of two U1A proteins bind specifically to two internal loops in the 3' untranslated region (3' UTR) of its own ... More
(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum.
AuthorsBishop JE, Squier TC, Bigelow DJ, Inesi G
JournalBiochemistry
PubMed ID2971396
Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface ... More
Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies.
AuthorsIngraham RH, Swenson CA
JournalBiochemistry
PubMed ID4074690
The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM ethylenediaminetetraacetic acid, a ... More
High hydrostatic pressure induces the dissociation of cpn60 tetradecamers and reveals a plasticity of the monomers.
AuthorsGorovits B, Raman CS, Horowitz PM
JournalJ Biol Chem
PubMed ID7836434
Hydrostatic pressures up to 2 kbar have been used to form monomers from the 14-subunit oligomer of the chaperonin, Cpn60. The fluorescence of 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS), followed at high pressure, demonstrated an increase in hydrophobic exposure on dissociation. Cpn60 dissociated with first order kinetics. The transition occurred between 1.3 ... More
Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells.
AuthorsOlmsted JB, Stemple DL, Saxton WM, Neighbors BW, McIntosh JR
JournalJ Cell Biol
PubMed ID2745548
To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP ... More
Phosphorylation-dependent changes in the spatial relationship between Ca-ATPase polypeptide chains in sarcoplasmic reticulum membranes.
AuthorsBigelow DJ, Squier TC, Inesi G
JournalJ Biol Chem
PubMed ID1532393
In order to investigate possible structural changes associated with the coupling mechanisms of the Ca-ATPase in sarcoplasmic reticulum membranes, we have utilized fluorescence resonance energy transfer between spectroscopic probes covalently bound to different domains of the ATPase. Using time-correlated single photon counting, we have directly measured the energy transfer efficiency ... More
Metal ion-catalyzed nucleic Acid alkylation and fragmentation.
AuthorsBrowne KA
JournalJ Am Chem Soc
PubMed ID12095339
Nucleic acid microarrays are a growing technology in which high densities of known sequences are attached to a substrate in known locations (addressed). Hybridization of complementary sequences leads to a detectable signal such as an electrical impulse or fluorescence. This combination of sequence addressing, hybridization, and detection increases the efficiency ... More
Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity.
AuthorsHammarström P, Persson M, Owenius R, Lindgren M, Carlsson U
JournalJ Biol Chem
PubMed ID10811634
Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the ... More
Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer.
AuthorsHorowitz PM, Hua S, Gibbons DL
JournalJ Biol Chem
PubMed ID7829481
The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. ... More
Structural effects of substrate utilization on the adenosinetriphosphatase chains of sarcoplasmic reticulum.
AuthorsWatanabe T, Inesi G
JournalBiochemistry
PubMed ID6214269
Addition of ATP to suspensions of fragmented sarcoplasmic reticulum (SR) containing low concentrations of a detergent that does not by itself produce major vesicular disruption is followed by a transient reduction in turbidity accompanied by solubilization of the vesicles. The effect of ATP is Ca2+ dependent and proceeds in parallel ... More
Translocation and clustering of endosomes and lysosomes depends on microtubules.
AuthorsMatteoni R, Kreis TE
JournalJ Cell Biol
PubMed ID3308906
Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of ... More