Abstract:
:A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Kusumi A,Tsuji A,Murata M,Sako Y,Yoshizawa AC,Kagiwada S,Hayakawa T,Ohnishi Sdoi
10.1021/bi00240a024subject
Has Abstractpub_date
1991-07-02 00:00:00pages
6517-27issue
26eissn
0006-2960issn
1520-4995journal_volume
30pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Bovine liver rhodanese, which catalyzes the transfer of sulfur atoms between a variety of sulfur donor and sulfur acceptor substrates, is inhibited by metal cyanide complexes [Volini, M., Van Sweringen, B., & Chen, F.-Sh. (1978) Arch. Biochem. Biophys. 191, 205-215]. Crystallographic studies are described which reveal...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00281a026
更新日期:1983-06-07 00:00:00
abstract::G proteins play an important role in transmitting hormonal signals, and fluorescence techniques would be useful to study their cellular distribution and mechanisms. To prepare active fluorescent G protein Go/Gi or beta gamma subunits were reacted with fluorescein isothiocyanate (FITC) to label the alpha (F-alpha) and ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00060a035
更新日期:1993-03-09 00:00:00
abstract::Spectrin dimers interact weakly with F-actin under physiological solvent conditions (with an association constant of about 5 X 10(3) M-1 at 20 degrees C). In the presence of the membrane skeletal constituent, protein 4.1, strong binding is observed; an analysis of the profiles for formation of a ternary complex leads ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00314a027
更新日期:1984-09-11 00:00:00
abstract::tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051863d
更新日期:2006-01-17 00:00:00
abstract::Human phenylalanine hydroxylase is a liver-specific enzyme that catalyzes the conversion of phenylalanine to tyrosine. Absence of enzymatic activity results in phenylketonuria, a genetic disorder that causes development of severe mental retardation in untreated children. In this paper we report the cloning and structu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00352a001
更新日期:1986-02-25 00:00:00
abstract::Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterizat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00496a011
更新日期:1990-11-06 00:00:00
abstract::The monoamine transporter of the chromaffin granule membranes can be specifically labeled by the photoaffinity reagent 7-azido-8-[125I]iodoketanserin. The characteristics of the labeled protein have been investigated. Two-dimensional gel electrophoresis of the labeled membranes indicated a MW of about 70,000 and an is...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00122a012
更新日期:1992-02-25 00:00:00
abstract::RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972671o
更新日期:1998-05-19 00:00:00
abstract::Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991271t
更新日期:1999-10-05 00:00:00
abstract::Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H(4)B) or tetrahydrobiopterin analogues (5-methyl-H(4)B and 4-amino-H(4)B), and the ability of bound 5-methyl-H(4)B and 4-amino-H(4)B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiN...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035491p
更新日期:2003-11-18 00:00:00
abstract::The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9830359
更新日期:1999-06-22 00:00:00
abstract::The location of the sites of methylation of the methyl-accepting chemotaxis proteins (MCP) of Escherichia coli has been investigated by trypsin digestion and mapping of the peptides. The two principal MCPs, MCP I and MCP II, were found to have very similar methyl-labeled peptide patterns. Each produced three major spe...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00507a051
更新日期:1981-02-17 00:00:00
abstract::Dog serum and colostral immunoglobulin A (IgA) and free secretory component from colostrum were isolated using affinity chromatography. Both serum and colostral IgA showed similar susceptibility to reduction with dithiothreitol, but only colostral IgA released the additional subunit, bound secretory component. This re...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00684a010
更新日期:1975-07-01 00:00:00
abstract::Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanis...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049803f
更新日期:2004-07-06 00:00:00
abstract::The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entir...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00491a016
更新日期:1990-10-02 00:00:00
abstract::In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF'.DNAn+1.PPi and KF.DNAn+1PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P.A....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00234a002
更新日期:1991-05-21 00:00:00
abstract::DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the calcium-dependent protease activity from DNA polymerase delta and alpha. The purified enzyme migrates a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00304a003
更新日期:1984-04-24 00:00:00
abstract::The nuclei and chromatin of rat liver contain three major proteins reacting with diisopropyl fluorophosphate (DFP). The molecular weights of the three proteins determined by gel filtration in the presence of sodium dodecyl sulfate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 70000, 60000, and 250...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00657a019
更新日期:1976-06-15 00:00:00
abstract::PII proteins are sensors of alpha-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9011594
更新日期:2009-12-08 00:00:00
abstract::Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by ca...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001629h
更新日期:2000-11-14 00:00:00
abstract::In the DNA recognition mode of C(2)H(2)-type zinc fingers, the finger-finger connection region, consisting of the histidine spacing (HX(3-5)H) and linker, would be important for determining the orientation of the zinc finger domains. To clarify the influence of spacing between two ligand histidines in the DNA binding,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035999s
更新日期:2004-05-25 00:00:00
abstract::Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chy...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00216a026
更新日期:1991-01-15 00:00:00
abstract::Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-co...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00511a019
更新日期:1981-04-14 00:00:00
abstract::Lactoferrin (Lf) and serum transferrin (Tf) combine high-affinity iron binding with an ability to release this iron at reduced pH. Lf, however, retains iron to significantly lower pH than Tf, giving the two proteins distinct functional roles. In this paper, we compared the iron-release profiles for human Lf, Tf, and t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0001224
更新日期:2000-06-06 00:00:00
abstract::Understanding the process of amyloidogenesis is important for the future treatment of misfolding-based diseases, such as Alzheimer's, spongiform encephalopathies, and other important disorders affecting humans. In this work, we have used one of the best-characterized models for folding and misfolding, the activation d...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi900960s
更新日期:2009-11-10 00:00:00
abstract::Despite a lack of convincing in vitro evidence and a number of sound refutations, it is widely accepted that renalase is an enzyme unique to animals that catalyzes the oxidative degradation of catecholamines in blood in order to lower vascular tone. Very recently, we identified isomers of β-NAD(P)H as substrates for r...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00451
更新日期:2015-06-23 00:00:00
abstract::Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi012077l
更新日期:2002-04-30 00:00:00
abstract::Silk fibroin exists in a number of different states, such as silk I and silk II, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the fac...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049344i
更新日期:2004-09-07 00:00:00
abstract::Because oxygen and selenium are in the same group (Family VI) in the periodic table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on the structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucle...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi060455m
更新日期:2006-07-25 00:00:00
abstract::The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049131t
更新日期:2004-10-05 00:00:00