Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We designed molecular beacons to detect a point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a mutation that has been related to an increased risk for cardiovascular disease and neural tube defects. The application of molecular beacons enables fast, semiautomated, ... More
Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.
AuthorsDischer DE, Winardi R, Schischmanoff PO, Parra M, Conboy JG, Mohandas N
JournalJ Cell Biol
PubMed ID7642705
Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB ... More
Rapid binding of synapsin I to F- and G-actin. A study using fluorescence resonance energy transfer.
AuthorsCeccaldi PE, Benfenati F, Chieregatti E, Greengard P, Valtorta F
JournalFEBS Lett
PubMed ID8365471
Synapsin I is a nerve terminal phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent manner. By using fluorescence resonance energy transfer between purified components labeled with fluorescent probes, we now show that the binding of synapsin I to actin is a rapid phenomenon. Binding of synapsin I ... More
Fluorescence energy transfers between points in acto-subfragment-1 rigor complex.
AuthorsMiki M, Wahl P
JournalBiochim Biophys Acta
PubMed ID6487641
Fluorescence energy transfer was measured by time-resolved and steady-state fluorimetry in order to investigate the spatial relationships between the nucleotide binding site of actin, the Cys-373 residue of actin, and the SH1 of myosin subfragment-1 in the rigor complex of acto-subfragment-1. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to the Cys-373 of actin or ... More
Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers.
AuthorsAmato PA, Taylor DL
JournalJ Cell Biol
PubMed ID3949874
'The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no ... More
Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases.
AuthorsSun QA, Wu Y, Zappacosta F, Jeang KT, Lee BJ, Hatfield DL, Gladyshev VN
JournalJ Biol Chem
PubMed ID10455115
'The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. ... More
Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.
AuthorsTetin SYu Mantulin WW, Denzin LK, Weidner KM, Voss EW
JournalBiochemistry
PubMed ID1457402
'This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major ... More
Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion.
AuthorsChiarugi P, Pani G, Giannoni E, Taddei L, Colavitti R, Raugei G, Symons M, Borrello S, Galeotti T, Ramponi G
JournalJ Cell Biol
PubMed ID12796479
'Signal transduction by reactive oxygen species (ROS; "redox signaling") has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a ... More
Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.
AuthorsSchedlich LJ, Le Page SL, Firth SM, Briggs LJ, Jans DA, Baxter RC
JournalJ Biol Chem
PubMed ID10811646
'Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions ... More
Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.
AuthorsJohnson CS, McKenna NM, Wang Y
JournalJ Cell Biol
PubMed ID3058721
'Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested ... More
Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.
AuthorsLin SH, Harzelrig JB, Cheung HC
JournalBiophys J
PubMed ID8274637
'The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was ... More
Microinjection of nonmuscle and smooth muscle caldesmon into fibroblasts and muscle cells.
AuthorsYamakita Y, Yamashiro S, Matsumura F
JournalJ Cell Biol
PubMed ID2277070
'Caldesmon is present in a high molecular mass form in smooth muscle and predominantly in a low molecular mass form in nonmuscle cells. Their biochemical properties are very similar. To examine whether these two forms of caldesmon behave differently in cultured cells, we microinjected fluorescently labeled smooth muscle and nonmuscle ... More
Energy transfer analysis of Fos-Jun dimerization and DNA binding.
AuthorsPatel LR, Curran T, Kerppola TK
JournalProc Natl Acad Sci U S A
PubMed ID8041795
'The protooncogenes fos and jun encode proteins that bind to DNA as dimeric complexes and regulate gene expression. Protein dimerization is mediated by a leucine zipper and results in juxtaposition of regions of each protein rich in basic amino acids that comprise a bimolecular DNA binding domain. We have developed ... More
Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin.
AuthorsAguirre R, Gonsoulin F, Cheung HC
JournalBiochemistry
PubMed ID3801396
'Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], ... More
p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.
AuthorsJans DA, Ackermann MJ, Bischoff JR, Beach DH, Peters R
JournalJ Cell Biol
PubMed ID1659575
'The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the ... More
Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.
AuthorsSaraswat LD, Pastra-Landis SC, Lowey S
JournalJ Biol Chem
PubMed ID1400421
'The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine ... More
Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site.
AuthorsGriep MA, Mesman TN
JournalBioconjug Chem
PubMed ID8608179
'Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase''s zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase ... More
The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations.
AuthorsNyitrai M, Hild G, Belágyi J, Somogyi B
JournalJ Biol Chem
PubMed ID10224049
'The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f'', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility ... More
Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.
AuthorsBark SJ, Hahn KM
JournalMethods
PubMed ID10720464
'When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to ... More
Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.
AuthorsBorejdo J, Ushakov DS, Akopova I
JournalBiophys J
PubMed ID12023239
'Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the ... More
Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.
'The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates ... More
A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.
AuthorsIsaacs BS, Husten EJ, Esmon CT, Johnson AE
JournalBiochemistry
PubMed ID3768326
'The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not ... More
Pyridoxal kinase. Structure and function.
AuthorsChurchich JE, Kim YT
JournalAnn N Y Acad Sci
PubMed ID2162645
'Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by HPLC and used for binding studies with ATP and pyridoxal analogues. The spectroscopic properties of ... More
Delivery of oligonucleotides into mammalian cells by anionic peptides: comparison between monomeric and dimeric peptides.
AuthorsFreulon I, Roche AC, Monsigny M, Mayer R
JournalBiochem J
PubMed ID11237872
'The use of antisense oligonucleotides as putative therapeutic agents is limited by their poor delivery into the cytosol and/or the nucleus because they are not able to efficiently cross lipid bilayers. To circumvent this pitfall, anionic amphipathic peptides derived from the influenza virus fusogenic peptide have been used to destabilize ... More
Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.
AuthorsKekic M, Huang W, Moens PD, Hambly BD, dos Remedios CG
JournalBiophys J
PubMed ID8804587
'We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) ... 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
Fluorescent modification of the cysteine 202 residue of Escherichia coli transcription termination factor rho.
AuthorsSeifried SE, Wang Y, von Hippel PH
JournalJ Biol Chem
PubMed ID2458348
'The lone cysteine residue (Cys-202) of transcription termination factor rho has been modified with the sulfhydryl-specific dyes 5-iodoacetamidofluorescein and 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid. Labeling with both dyes is specific for the Cys-202 residue and is at least 90% complete. Rho protein is an RNA-dependent ATPase and exists as a hexamer of identical ... More
Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers.
AuthorsBorejdo J, Assulin O
JournalBiochemistry
PubMed ID7000187
'The binding of fluorescently labeled heavy meromyosin (HMM) and heavy meromyosin subfragment-1 (S-1) to thin filaments of myofibrils and of rabbit psoas muscle fibers was measured under conditions of rigor and contraction. The fragments diffused rapidly into the myofibrillar space and bound specifically to the thin filaments. The fragments bound ... More
Endogenous muscle lectin inhibits myoblast adhesion to laminin.
AuthorsCooper DN, Massa SM, Barondes SH
JournalJ Cell Biol
PubMed ID1955484
'L-14, a dimeric lactose-binding lectin with subunits of 14 kD, is expressed in a wide range of vertebrate tissues. Several functions have been postulated for this lectin, but definitive evidence for a specific biological role has been elusive. In muscle, L-14 is secreted during differentiation and accumulates with laminin in ... More
Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.
AuthorsChantler PD, Tao T
JournalJ Mol Biol
PubMed ID3820308
'Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified ... 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
Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB).
AuthorsGrossman SH
JournalBiochemistry
PubMed ID6652070
'The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5''-(iodoacetamido)fluorescein (5''-IAF). Spectral analyses demonstrated that 1.8 mol ... More
Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.
AuthorsPutilina T, Skouri-Panet F, Prat K, Lubsen NH, Tardieu A
JournalJ Biol Chem
PubMed ID12562766
'The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for ... More
Engineered cysteine mutants of myosin light chain 2. Fluorescent analogues for structural studies.
AuthorsSaraswat LD, Lowey S
JournalJ Biol Chem
PubMed ID1918082
'Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine ... More
Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit.
AuthorsWensel TG, Stryer L
JournalBiochemistry
PubMed ID2158346
'The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme ... More
Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence.
AuthorsAbrahamson JK, Laue TM, Miller DL, Johnson AE
JournalBiochemistry
PubMed ID3888260
'We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the ... More
Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence.
AuthorsBurghardt TP, Cruz-Walker AR, Park S, Ajtai K
JournalBiochemistry
PubMed ID11294650
'Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the ... More
Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation.
AuthorsPhan BC, Cheung P, Stafford WF, Reisler E
JournalBiophys Chem
PubMed ID8672721
'Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi state while the latter resembles more the S1.ATP state. In ... More
Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.
AuthorsCeccaldi PE, Grohovaz F, Benfenati F, Chieregatti E, Greengard P, Valtorta F
JournalJ Cell Biol
PubMed ID7876313
'Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles ... More
Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization.
AuthorsWan QH, Le XC
JournalAnal Chem
PubMed ID11101235
'Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to ... More
Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.
'Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, ... More
'Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share ... More
Conformational transitions and change translocation by the Na,K pump: comparison of optical and electrical transients elicited by ATP-concentration jumps.
AuthorsStürmer W, Apell HJ, Wuddel I, Läuger P
JournalJ Membr Biol
PubMed ID2552127
'The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (approximately 8000 microm(-2)) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive ... More
Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.
'Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior ... More
Estimation of the distance change between cysteine-457 and the nucleotide binding site when sodium pump changes conformation from E1 to E2 by fluorescence energy transfer measurements.
AuthorsLin SH, Faller LD
JournalBiochemistry
PubMed ID8679600
'The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the alpha-subunit. The donor (5''-(iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2''(or 3'')-O-(trinitrophenyl)adenosine 5''-triphosphate) ... More
Evidence for allosteric linkage between exosites 1 and 2 of thrombin.
AuthorsFredenburgh JC, Stafford AR, Weitz JI
JournalJ Biol Chem
PubMed ID9325262
'Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and ... More
A method for incorporating macromolecules into adherent cells.
AuthorsMcNeil PL, Murphy RF, Lanni F, Taylor DL
JournalJ Cell Biol
PubMed ID6201494
'We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold ... More
Molecular weight of human high-molecular-weight kininogen light chain by equilibrium sedimentation in an air-driven ultracentrifuge.
AuthorsBock PE, Halvorson HR
JournalAnal Biochem
PubMed ID6670737
'High-molecular-weight kininogen, a nonenzymatic glycoprotein of the intrinsic blood coagulation system, is proteolytically cleaved by kallikrein as an early event in the activation of this system. The light chain of cleaved kininogen retains the ability to form specific noncovalent complexes with prekallikrein and factor XI, other members of this system. ... More
Velocity of movement of actin filaments in in vitro motility assay. Measured by fluorescence correlation spectroscopy.
AuthorsBorejdo J, Burlacu S
JournalBiophys J
PubMed ID1534696
'We have measured the velocity of actin filaments in in vitro motility assay by fluorescence correlation spectroscopy. In this method, one measures fluctuations in the number of filaments in an open sample volume. The number of filaments was calculated from measurements of fluorescence of rhodamine-phalloidin bound to F-actin. Sample volume ... More
Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein.
AuthorsKapakos JG, Steinberg M
JournalJ Biol Chem
PubMed ID3003093
'The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5''-yl imidodiphosphate ... More
Effects of villin on the polymerization and subunit exchange of actin.
AuthorsWang Y, Bonder EM, Mooseker MS, Taylor DL
JournalCell Motil
PubMed ID6883468
'We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. ... More
Luminescent/paramagnetic probes of order and orientation in biological assemblies: the transformation of luminescent probes into pi-radicals by photochemical reduction within the contractile apparatus.
AuthorsBurghardt TP, Ajtai K
JournalAdv Exp Med Biol
PubMed ID7509109
'Several unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction the quinoidal structure of the xanthene ring is transformed into a semiquinone and a pi-radical is formed having a characteristic electron paramagnetic ... More
5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover.
AuthorsKapakos JG, Steinberg M
JournalJ Biol Chem
PubMed ID3003094
'The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% ... More
Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.
AuthorsKhan AR, Williams KA, Boggs JM, Deber CM
JournalBiochemistry
PubMed ID7547983
'The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein ... More
Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.
AuthorsAnsorge W, Sproat B, Stegemann J, Schwager C, Zenke M
JournalNucleic Acids Res
PubMed ID3588303
'A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 ... More
Mobility of cytoplasmic and membrane-associated actin in living cells.
AuthorsWang YL, Lanni F, McNeil PL, Ware BR, Taylor DL
JournalProc Natl Acad Sci U S A
PubMed ID6956883
'We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that ... More
Diffusion-enhanced energy transfer investigation of histone H5 in chromatin with a fluorescently-labelled antibody fragment Fab'.
AuthorsSarlet G, Muller S, Houssier C
JournalJ Biomol Struct Dyn
PubMed ID1418745
'The location of chicken erythrocyte H5 histone relative to the axis the 30 nm chromatin fibre axis has been investigated by diffusion-enhanced energy transfer. In this investigation, a neutral lanthanide chelate as donor and a fluorescent probe specific to H5 as acceptor have been used. The acceptor probe consists of ... More
Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer.
AuthorsPalm T, Sale K, Brown L, Li H, Hambly B, Fajer PG
JournalBiochemistry
PubMed ID10529172
'The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science ... More
Wavelength-shifting molecular beacons.
AuthorsTyagi S, Marras SA, Kramer FR
JournalNat Biotechnol
PubMed ID11062440
'We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light ... More
Characterization of an active, fluorescein-labelled kinesin.
AuthorsMarya PK, Fraylich PE, Eagles PA
JournalEur J Biochem
PubMed ID2146115
'Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a ... More
Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells.
AuthorsDeBiasio R, Bright GR, Ernst LA, Waggoner AS, Taylor DL
JournalJ Cell Biol
PubMed ID2444600
'Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined ... More
Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.
'The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, ... More
Segmental motion and rotational diffusion of the Ca2+-translocating adenosine triphosphatase of sarcoplasmic reticulum, measured by time-resolved phosphorescence depolarization.
AuthorsSpeirs A, Moore CH, Boxer DH, Garland PB
JournalBiochem J
PubMed ID6137210
'We studied the rotational mobility of the Ca2+ + Mg2+-activated ATPase in skeletal-muscle sarcoplasmic-reticulum vesicles, using time-resolved measurements of the depolarization of laser-flash-excited phosphorescence of the extrinsic triplet probe erythrosin. Our results are in general agreement with those of others [Bürkli & Cherry (1981) Biochemistry 20, 138-145] obtained by linear ... More
Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.
AuthorsWang YL, Taylor DL
JournalCell
PubMed ID6101197
'Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We ... More
Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification.
AuthorsMeunier L, Bourgerie S, Mayer R, Roche AC, Monsigny M
JournalBioconjug Chem
PubMed ID10077469
'A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6''-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated ... More
Kinetics of histone endocytosis in Chinese hamster ovary cells. A flow cytofluorometric analysis.
AuthorsMurphy RF, Jorgensen ED, Cantor CR
JournalJ Biol Chem
PubMed ID7056738
'The endocytosis of histones by cultured cells was examined by flow cytofluorometry. Monolayer cultures of Chinese hamster ovary cells were incubated with fluorescein-labeled histone for various periods of time and then trypsinized to remove surface-bound protein. Internalization followed first order kinetics with a half-time of 45 min, and was linear ... More
Resonance energy transfer: methods and applications.
AuthorsWu P, Brand L
JournalAnal Biochem
PubMed ID8053542
'Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 A and is thus suitable for investigating spatial relationships of interest in biochemistry. The information available from energy transfer studies has been enhanced by the ... More
Detection of actin assembly by fluorescence energy transfer.
AuthorsTaylor DL, Reidler J, Spudich JA, Stryer L
JournalJ Cell Biol
PubMed ID6894758
'Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole ... More
Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease.
'The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme ... More
The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions.
'Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the ... More
Binding and distribution of fluorescently labeled filamin in permeabilized and living cells.
AuthorsMittal B, Sanger JM, Sanger JW
JournalCell Motil Cytoskeleton
PubMed ID3690693
'This study reports the first development of a fluorescently labeled filamin. Smooth muscle filamin was labeled with fluorescent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bound to the Z bands of isolated cross-striated myofibrils ... More
Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin.
AuthorsKim E, Bobkova E, Miller CJ, Orlova A, Hegyi G, Egelman EH, Muhlrad A, Reisler E
JournalBiochemistry
PubMed ID9922146
'Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and ... More
Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas.
AuthorsTaylor DL, Wang YL, Heiple JM
JournalJ Cell Biol
PubMed ID6893200
'The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein-labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same ... More
Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.
AuthorsBühler R, Stürmer W, Apell HJ, Läuger P
JournalJ Membr Biol
PubMed ID1652643
'Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). ... More
A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import.
AuthorsXiao CY, Hübner S, Elliot RM, Caon A, Jans DA
JournalJ Biol Chem
PubMed ID8626446
'The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, ... More
Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase.
AuthorsTyson PA, Steinberg M, Wallick ET, Kirley TL
JournalJ Biol Chem
PubMed ID2536022
'5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived ... More
Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase.
AuthorsKirley TL, Lane LK, Wallick ET
JournalJ Biol Chem
PubMed ID3007461
'Ellman''s reagent 5,5''-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be ... More
Tertiary structural changes in the cleft containing the ATP sensitive tryptophan and reactive thiol are consistent with pivoting of the myosin heavy chain at Gly699.
AuthorsBurghardt TP, Garamszegi SP, Park S, Ajtai K
JournalBiochemistry
PubMed ID9609697
'The conformation of myosin subfragment 1 (S1) in the vicinity of the ATP sensitive tryptophan (Trp510) and the highly reactive thiol (SH1), both residing in the "probe-binding" cleft at the junction of the catalytic and lever arm domains, was studied to ascertain its role in the mechanism of energy transduction ... More
Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis.
AuthorsCarru C, Deiana L, Sotgia S, Pes GM, Zinellu A
JournalElectrophoresis
PubMed ID15004850
'High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved ... More
Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.
AuthorsWang YL, Taylor DL
JournalJ Histochem Cytochem
PubMed ID6107318
'The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein ... More
Determination of the fractal dimension of membrane protein aggregates using fluorescence energy transfer.
AuthorsDewey TG, Datta MM
JournalBiophys J
PubMed ID2528385
'It is demonstrated that fluorescence resonance energy transfer may be used to determine the fractal dimension of aggregates of membrane-bound proteins. Theoretical and experimental results are presented for two different experimental designs: energy transfer between proteins and energy transfer from lipids to proteins. For energy transfer between proteins the lattice ... More
Modification of Escherichia coli ribosomes with the fluorescent reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Identification of derivatized L31' and studies on its intraribosomal properties.
AuthorsHanas JS, Simpson MV
JournalBiochemistry
PubMed ID3910100
'N-[[(Iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS) is a fluorescent reagent which reacts covalently with the free thiol groups of proteins. When the reagent is reacted with the Escherichia coli ribosome under mild conditions, gel electrophoresis shows modification of predominantly two proteins, S18 and L31'', which become labeled to an equal extent. When the ... More
Self-quenched fluorogenic protein substrates for the detection of cathepsin D and other protease activities.
AuthorsAshcom JD, Jacobson LA
JournalAnal Biochem
PubMed ID2662807
'Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including ... More
Rotational dynamics of erythrocyte spectrin.
AuthorsLearmonth RP, Woodhouse AG, Sawyer WH
JournalBiochim Biophys Acta
PubMed ID2597682
'The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere ... More
Binding of heavy-chain and essential light-chain 1 of S1 to actin depends on the degree of saturation of F-actin filaments with S1.
AuthorsAndreev OA, Borejdo J
JournalBiochemistry
PubMed ID7578092
'The interaction of heavy-chain isoforms of myosin subfragment-1 with actin was examined by cross-linking with carbodiimide (EDC). The heavy chain of S1 could be cross-linked to a single actin molecule through sites on either 20 or 50 kDa proteolytic domains, resulting in complexes which migrated in an 8% polyacrylamide gel ... More
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Studies using a fluorescent analogue of kinesin.
AuthorsMarya PK, Fraylich PE, Flood CR, Rao R, Eagles PA
JournalJ Cell Sci Suppl
PubMed ID1885652
'The microtubule motor protein kinesin has been conjugated with 5-iodoacetamido fluorescein (5-IAF). The analogue, AF-kinesin, supports organelle motility and the movement of microtubules.'
Distribution of actin in spreading macrophages: a comparative study on living and fixed cells.
AuthorsAmato PA, Unanue ER, Taylor DL
JournalJ Cell Biol
PubMed ID6339523
Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification.
AuthorsWehland J, Weber K
JournalExp Cell Res
PubMed ID6892899
Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment.
AuthorsFurey WS, Joyce CM, Osborne MA, Klenerman D, Peliska JA, Balasubramanian S
JournalBiochemistry
PubMed ID9485450
'Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors ... More
Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states.
AuthorsTrayer HR, Trayer IP
JournalBiochemistry
PubMed ID2972314
'Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography ... More
Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite.
AuthorsTiago T, Simão S, Aureliano M, Martín-Romero FJ, Gutiérrez-Merino C
JournalBiochemistry
PubMed ID16533063
'Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced ... More
Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin.
AuthorsDasGupta G, White J, Cheung P, Reisler E
JournalBiochemistry
PubMed ID2252908
'The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by ... More
Plasma D-penicillamine redox state evaluation by capillary electrophoresis with laser-induced fluorescence.
AuthorsZinellu A, Carru C, Sotgia S, Deiana L
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID15063339
'D-Penicillamine (D-Pen) is a thiol drug used in the treatment of Wilson''s disease, rheumatoid arthritis, metal intoxication and cystinuria. We have recently described a new capillary electrophoresis (CE) method to measure physiological thiols, in which separation of total plasma homocysteine, cysteine, cysteinylglycine, glutathione is achieved using the organic base N-methyl-D-glucamine ... More
Probe dependence of correlation times in heme-free extrinsically labeled human hemoglobin.
AuthorsSassaroli M, Kowalczyk J, Bucci E
JournalArch Biochem Biophys
PubMed ID3800389
'Human heme-free hemoglobin was labeled at beta 93 with either N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate (AEDANS) or fluorescein iodoacetamide (FIA), at beta 1 with pyridoxal-5-phosphate (PLP) and at the heme pocket with anilinonaphthalene-8-sulfonate (ANS). The correlation times associated with these probes ranged from approximately 12 ns for FIA and AEDANS to nearly 20 ns ... More
The rate of MgADP binding to and dissociation from acto-S1.
AuthorsBorejdo J, Ando T, Burghardt TP
JournalBiochim Biophys Acta
PubMed ID3872135
'The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent ... More
Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates.
AuthorsJonsson T, Waldburger CD, Sauer RT
JournalBiochemistry
PubMed ID8664269
'Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Arc dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Arc variants labeled with fluorescence acceptor or donor groups and used energy ... More
Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
AuthorsSimard JR, Getlik M, Grütter C, Pawar V, Wulfert S, Rabiller M, Rauh D,
JournalJ Am Chem Soc
PubMed ID19572644
'Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less ... More
Flow cytofluorometric analysis of insulin binding and internalization by Swiss 3T3 cells.
AuthorsMurphy RF, Powers S, Verderame M, Cantor CR, Pollack R
JournalCytometry
PubMed ID6804197
'The binding of a fluorescein-isothiocyanate derivative of insulin to Swiss 3T3 cells was measured by flow cytometry. The kinetics of the subsequent internalization were also measured; at a concentration of 1 microM labeled insulin approximately 25% of the internalization was insulin-specific. The kinetics of endocytosis were contrasted to those of ... More
Interactions between a viral protease and cystatins.
AuthorsKorant B, Towatari T, Kelley M, Brzin J, Lenarcic B, Turk V
JournalBiol Chem Hoppe Seyler
PubMed ID3060140
'The interactions of two cystatins with a viral cysteine protease were studied using several types of assays. Complex formation between the protease and inhibitor was directly demonstrated using a gel retardation assay. It was also shown that the formation of enzyme inhibitor complexes could occur after first binding either the ... More