Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1.
AuthorsMaruta S, Mizukura Y, Chaen S
JournalJ Biochem (Tokyo)
PubMed ID12038988
'A new fluorescent ribose-modified ATP analogue, 2''(3'')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and ... More
Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids.
AuthorsMartin OC, Pagano RE
JournalAnal Biochem
PubMed ID3812987
'We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3-diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBD-aminocaproyl (C6-NBD) or NBD-aminododecanoyl (C12-NBD) acyl chains were synthesized and analyzed by the following HPLC methods. An isocratic normal-phase method permitted resolution of ... More
Sorting of sphingolipids in the endocytic pathway of HT29 cells.
AuthorsKok JW, Babia T, Hoekstra D
JournalJ Cell Biol
PubMed ID2071671
'The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues ... More
Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis.
AuthorsKok JW, Eskelinen S, Hoekstra K, Hoekstra D
JournalProc Natl Acad Sci U S A
PubMed ID2690077
'To examine the (intra)cellular fate of a glycolipid, normally residing at the cell surface, a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsp hingosine (C6-NBD-glucosylceramide), was inserted into the plasma membrane of baby hamster kidney cells at low temperature. Upon warming the cells to 37 degrees C, part of the glycolipid analog was ... More
Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids.
AuthorsRosenwald AG, Pagano RE, Raviv Y
JournalJ Biol Chem
PubMed ID2033068
'We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently ... More
Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages.
AuthorsTanaka Y, Schroit AJ
JournalJ Biol Chem
PubMed ID6885820
'The interaction of macrophages with red blood cells (RBC) displaying phosphatidylserine (PS) in their surface membranes was investigated after the transfer of an exogenously supplied fluorescent lipid analog to the RBC. Nonfluorescent (quenched) lipid vesicles were formed by ultrasonication from 1-acyl-2-[(N-4-nitro-benzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidyl-serine (NBD-PS) or 1-acyl-2[(N-4-nitrobenzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidylcholine (NBD-PC). The interaction of ... More
Fluorescence study of a derivatized diacylglycerol incorporated in model membranes.
AuthorsPrieto MJ, Castanho M, Coutinho A, Ortiz A, Aranda FJ, Gómez-Fernández JC
JournalChem Phys Lipids
PubMed ID8200058
'A fluorescence study of a diacylglycerol derivatized with the n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) chromophore (NBD) was carried out. Fluorescence self-quenching was observed for this probe in lecithin model membranes due to collisional interaction rather than to an aggregational behaviour of the probe. The efficient energy migration (Ro = 28 A) of the NBD ... More
In vitro fusion of endosomes following receptor-mediated endocytosis.
AuthorsDiaz R, Mayorga L, Stahl P
JournalJ Biol Chem
PubMed ID3360775
'Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment ... More
Fluorescence detection in automated DNA sequence analysis.
AuthorsSmith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE
JournalNature
PubMed ID3713851
'We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific ... More
Detection of protein mediated glycosphingolipid clustering by the use of resonance energy transfer between fluorescent labelled lipids. A method established by applying the system ganglioside GM1 and cholera toxin B subunit.
AuthorsAntes P, Schwarzmann G, Sandhoff K
JournalChem Phys Lipids
PubMed ID1468126
'Glycosphingolipids labelled in the ceramide moiety with 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH) or 6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)aminohexanoic acid (NBD) were incorporated into small unilamellar lecithin liposomes. They were used in resonance energy transfer (RET) experiments between the donor fluorophore DPH and the acceptor NBD to study glycosphingolipid distribution. In pure lecithin liposomes the fluorescent derivatives ... More
Translocation of fluorescent ether phospholipid, but not its diacyl counterpart, after insertion in plasma membranes of control and plasmalogen-deficient fibroblasts.
AuthorsVan der Veer E, Van der Weide D, Heijmans HS, Hoekstra D
JournalBiochim Biophys Acta
PubMed ID8452863
'Fluorescently labelled ether phospholipid (1-O-alkyl/alkenyl-2-acyl- glycerophosphocholine) readily internalizes at low temperatures (2 degrees C) after insertion into the plasma membrane of cultured fibroblasts. This fate differs markedly from that of its diacyl phospholipid analogue, which remains associated with the plasma membrane under similar conditions. Analysis by thin-layer chromatography reveals that ... More
Synthesis and characterization of fluorescent neutral lipid analogs containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-aminohexanoic acid.
AuthorsMcIntyre JC, Watson D, Sleight RG
JournalChem Phys Lipids
PubMed ID8111933
'The synthesis, identification and characterization of neutral lipid analogs containing N-(7-nitro-2,1,3-benzoxadiazoi-4-yl)-aminocaproic acid are reported. The acyl-imidazole derivative of the fluorescent fatty acid was used to esterify L-alpha-glycerophosphorylcholine. Fluorescent phosphatidylcholines were converted to the corresponding diacylglycerols by phospholipase C digestion. Triacylglycerols were formed by esterification with either fluorescent fatty acid-imidazole or ... More
Synthesis and properties of a fluorescent derivative of phosphatidylcholine.
AuthorsMonti JA, Christian ST, Shaw WA, Finley WH
JournalLife Sci
PubMed ID895370
Spectroscopic studies on invertebrate myosins and light chains.
AuthorsChantler PD, Szent-Györgyi AG
JournalBiochemistry
PubMed ID215199
Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction.
AuthorsDraper DE
JournalNucleic Acids Res
PubMed ID6198634
Bisulfite catalyzes transamination of cytidine at the N4 position; the suitability of this reaction for attaching reporter groups to selected cytidine residues in RNA molecules has been investigated. Poly(C) is nearly quantitatively converted to the poly (N4 aminoethyl-C) derivative after 3 hrs at 42 degrees C with ethylene diamine (pK1 ... More
Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid.
AuthorsLongmuir KJ, Martin OC, Pagano RE
JournalChem Phys Lipids
PubMed ID4006033
Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate ... More
Time-resolved fluorescence of nitrobenzoxadiazole-aminohexanoic acid: effect of intermolecular hydrogen-bonding on non-radiative decay.
AuthorsLin S, Struve WS
JournalPhotochem Photobiol
PubMed ID1784635
The fluorescence kinetics of the nitrobenzoxadiazole (NBD) chromophore were studied at low concentrations in solvents with varying polarity and hydrogen-bonding donor strength. The emission decay was essentially single exponential in all solvents studied. While the absorption and fluorescence solvatochromism is determined largely by the solvent polarity, the S1 state decay ... More
Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles.
AuthorsDao HN, McIntyre JC, Sleight RG
JournalAnal Biochem
PubMed ID1888035
A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid ... More
Relationship between lateral diffusion, collision frequency, and electron transfer of mitochondrial inner membrane oxidation-reduction components.
Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-c1), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and ... More
On-the-fly frequency-domain fluorescence lifetime detection in capillary electrophoresis.
AuthorsLi LC, McGown LB
JournalAnal Chem
PubMed ID8794914
On-the-fly fluorescence lifetime detection in capillary electrophoresis (CE) is demonstrated. Virtually continuous detection is achieved by interfacing a commercial CE instrument with a commercial, multiharmonic Fourier transform lifetime fluorometer (MHF). The CE capillary cartridge was modified to allow the capillary to pass through a specially constructed capillary column mount capable ... More
Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.
AuthorsEskelinen S, Kok JW, Sormunen R, Hoekstra D
JournalEur J Cell Biol
PubMed ID1802708
We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell ... More
Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion.
AuthorsGoennenwein S, Tanaka M, Hu B, Moroder L, Sackmann E
JournalBiophys J
PubMed ID12829518
Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, ... More
Polarized sphingolipid transport from the subapical compartment: evidence for distinct sphingolipid domains.
Authorsvan IJzendoorn SC, Hoekstra D
JournalMol Biol Cell
PubMed ID10512879
In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains ... More
NBD-5-acylcholine: fluorescent analog of acetylcholine and agonist at the neuromuscular junction.
AuthorsJürss R, Prinz H, Maelicke A
JournalProc Natl Acad Sci U S A
PubMed ID312497
We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is ... More
An investigation into the glycolipid metabolism of alpha-N-acetylgalactosaminidase-deficient fibroblasts using native and artificial glycolipids.
AuthorsKlima B, Pohlentz G, Schindler D, Egge H
JournalBiol Chem Hoppe Seyler
PubMed ID1418679
Deficient activity of lysosomal alpha-N-acetylgalactosaminidase represents a recently recognized lysosomal disorder whose neurologic manifestation in infancy is infantile neuroaxonal dystrophy. The lysosomal enzyme defect, inherited as an autosomal recessive trait, was first identified in the two brothers, GD and BD. Metabolic modification of glycolipids with terminal alpha-GalNAc was studied in ... More
Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes.
AuthorsChattopadhyay A, London E
JournalBiochim Biophys Acta
PubMed ID3337814
The spectroscopic and ionization properties of various lipids labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group have been studied in model membranes using fluorescence, absorbance and electrophoretic mobility measurements. Electrophoretic measurements show that the NBD group is uncharged at neutral pH. However, at high pH, hydroxyl addition or deprotonation occurs with a ... More
Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.
AuthorsMayor S, Presley JF, Maxfield FR
JournalJ Cell Biol
PubMed ID8509447
A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane-associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, ... More
Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol.
AuthorsSchroeder F, Myers-Payne SC, Billheimer JT, Wood WG
JournalBiochemistry
PubMed ID7547928
Direct effects of ethanol on the interaction of cytosolic lipid transfer proteins with ligands are not known. In this study, recombinant liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2) were used in conjunction with a series of fluorescent fatty acid probe molecules to compare the relative dielectric ... More
Fluorophore environments in membrane-bound probes: a red edge excitation shift study.
AuthorsChattopadhyay A, Mukherjee S
JournalBiochemistry
PubMed ID8466919
A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the Red Edge Excitation Shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as ... More
Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.
AuthorsMilhiet PE, Giocondi MC, Crine P, Le Grimellec C, Roques BP, Garbay-Jaureguiberry C
JournalEur J Cell Biol
PubMed ID7720721
Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the ... More
Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites.
AuthorsOsiecki-Newman K, Fabbro D, Legler G, Desnick RJ, Grabowski GA
JournalBiochim Biophys Acta
PubMed ID2956992
Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of ... More
Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid storage disease fibroblasts.
AuthorsIgal RA, Coleman RA
JournalJ Biol Chem
PubMed ID8663220
Neutral lipid storage disease (NLSD) is an autosomal recessive disorder in which excess triacylglycerol (TG) accumulates in most cells. Although it has been hypothesized that the TG accumulation is caused by a functional defect in cytosolic lipase activity, we were able to expose TG hydrolysis in NLSD cells by using ... More
Bimodal regulation of ceramidase by interleukin-1beta. Implications for the regulation of cytochrome p450 2C11.
AuthorsNikolova-Karakashian M, Morgan ET, Alexander C, Liotta DC, Merrill AH
JournalJ Biol Chem
PubMed ID9228043
Interleukin 1beta (IL-1beta) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Cer has been proposed to play a role in the down-regulation of cytochrome P450 2C11 (CYP2C11) and induction of alpha1-acid glycoprotein (AGP) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. ... More
Sphingolipid transport to the apical plasma membrane domain in human hepatoma cells is controlled by PKC and PKA activity: a correlation with cell polarity in HepG2 cells.
AuthorsZegers MM, Hoekstra D
JournalJ Cell Biol
PubMed ID9230073
The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent ('direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes ... More
Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes.
AuthorsKok JW, Hoekstra K, Eskelinen S, Hoekstra D
JournalJ Cell Sci
PubMed ID1487494
Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's ... More
Fluorescence assay for phospholipid membrane asymmetry.
AuthorsMcIntyre JC, Sleight RG
JournalBiochemistry
PubMed ID1751498
Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were ... More
Effect of diacylglycerols on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2.
AuthorsZidovetzki R, Laptalo L, Crawford J
JournalBiochemistry
PubMed ID1510954
The effects of a series of diacylglycerols (DAGs) with varying acyl chain lengths and degree of unsaturation on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2 (PL-A2S) were studied using two lipid substrates: dipalmitoylphosphatidylcholine (DPPC) or bovine liver phosphatidylcholine (BL-PC). The activities of the phospholipases critically ... More
Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.
AuthorsKoval M, Pagano RE
JournalJ Cell Biol
PubMed ID2380243
We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells ... More
The mobility of a fluorescent ubiquinone in model lipid membranes. Relevance to mitochondrial electron transport.
AuthorsChazotte B, Wu ES, Hackenbrock CR
JournalBiochim Biophys Acta
PubMed ID2065063
The diffusion and location of a functional, fluorescent ubiquinone molecule, NBDHA-Q, were determined as a function of temperature using microscopic observation, fluorescence recovery after photobleaching and fluorescence spectroscopy in protein-free, pure-lipid dimyristoylphosphatidylcholine and dimyristoylphosphatidylcholine/cholesterol multibilayers. The data reveal that in a liquid-crystalline membrane (1) ubiquinone is highly mobile, (2) ubiquinone ... More
A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells.
AuthorsPagano RE, Martin OC
JournalBiochemistry
PubMed ID3166987
We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma ... More