Biochemical aspects of the visual process. XXII. Amino group modification in bovine rod photoreceptor membranes.
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Alcohol Oxidoreductases↗
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Results for “Dansyl Compounds”
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Interaction between erythrocyte membrane proteins and complement components. I. The role os -S-S linkages as revealed by two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis.
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Animals↗
Effects of ions on vesicles and phospholipid dispersions studied by polarization of fluorescence.
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Binding Sites↗
Absence of cell communication for fluorescein and dansylated amino acids in an electrotonic coupled cell system.
Quantitative evaluation of the diffusion process of sodium fluorescein and dansylated amino acids in the salivary gland of the larvae of Drosophila hydei reveals that the differences in specific permeability between the junctional and nonjunctional membranes, as found for small ions, do not apply to the fluorescent probes. There are no significant differences between the permeability properties for the different dansylated amino acids tested, and the same properties are found for sodium fluorescein.
Animals↗
Rapid preparative isolation of major erythrocyte membrane proteins using polyacrylamide gel electrophoresis in sodium dodecylsulfate.
1. We describe a simple method for preparative, sodium dodecylsulfate/polyacrylamide gel electrophoresis of the major proteins in human erythrocyte membranes. 2. The method is based on extraction of prestained proteins from gel slabs. Three different fluorescent dyes (o-phthalaldehyde, fluorescamine and 1-dimethyl-aminonaphthalene-5-sulfonylchloride) have been used for pretaining. The method allows separation of up to 75 mg membrane protein and isolation of mg quantities of all major erythrocyte ghost proteins, while preserving the high resolution of analytical polyacrylamide gel electrophoresis. 3. Yield depends on extraction conditions and the molecular weight of the proteins being eluted. It ranges from 43-48% for protein 1 (apparent mol. wt approx. 310000) and 72-78% for protein 3(apparent mol. wt 87 000-93 000) to 87-93% for protein 6 (apparent mol. wt 35 000). 4. The labile behaviour of the high molecular "spectrin" bands (bands 1 and 2) is described. Extraction at room temperature tends to split these proteins into products of lower molecular weight. In contrast, the minor protein components 2.1 and 2.2 tend to aggregate yielding components 1 and 2. 5. N-terminal amino acid analyses have been performed on proteins 1, 2, 3, 4A, 4B, 5 and 6. Each of these bands contains several N-terminals, most of which appear constant. Some additional N-terminal amino acids vary from one donor to the next.
Amino Acid Sequence↗
Labelling of sarcoplasmic reticulum membranes with 1-dimethylaminonaphthalene-5-sulfonyl chloride.
1. Sarcoplasmic reticulum membranes were labelled with 1-dimethylaminonaphtalene-5-sulfonyl chloride (DnsCl). Analyses of the dansylated membranes demonstrated that the most of the dye was associated with ATPase (ATP phosphohydrolase, EC 3.6.1.3) and phosphatidylethanolamine in the membranes. 2. Dansylation of the membranes could be performed without significant decrease in the ATPase activity. 3. Partial differentiation of fluorescence of Dns-phosphatidylethanolamine from that of Dns-ATPase could be achieved by changing excitation wavelength; Dns-ATPase emmitted in the shorter wavelength region, while Dns-phosphatidylethanolamine emmitted in the longer wavelength region. 4. Fluorescence polarization of the dye bound to the membranes indicated that both the ATPase and phosphatidylethanolamine were strongly immobilized in the membranes, while the ratio of freely rotating dye to the "frozen" dye bound to the ATPase was larger than that bound to the phosphatide.
Adenosine Triphosphatases↗
Interaction of a new fluorescent reagent with sulfhydryl groups of the (Na+ + K+)-stimulate ATPase.
The (Na+ + K+)-ATPase enzyme of rat brain microsomes can be reversibly inhibited by a new fluorescent sulfhydryl (SH) probe, dimethylaminoaphthalenecysteine-Hg+ (Dn-cys-Hg+). This reagent is more reactive than N-ethylamaleimide (MalNEt) toward membrane sulfhydryl groups. A procedure using the two SH reagents sequentially seems to permit a more selective labelling of the SH groups involved in the (Na+ + K+)-ATPase than is possible by using MalNEt alone. Brain microsomes treated by this method incorporate the fluorescent label within or near the active site of the enzyme. When the probe was bound a blue shift of its fluorescence emission maximum (from 540 to 495 nm) and a 20-fold increase in relative fluorescence occurred. This indicates that the Dn moiety is within a very non-polar region of the membrane.
Adenosine Triphosphatases↗
Fluorescence temperature jump relaxations of dansylphosphatidylethanolamine in aqueous dispersions of dipalmitoylphosphatidylcholine during the gel to liquid-crystal transition.
The change of fluorescence of the probe dansylphosphatidylethanolamine embedded in multilamellar liposomes of dipalmitoylphosphatidylcholine was used to investigate the dynamics of the thermotropic gel to liquid-crystalline phase transition by use of the temperature-jump technique. The results are discussed and compared to published observations on the same system in which the phenomenon was reported by turbidity changes.
Dansyl Compounds↗
Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels.
A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.
Antibodies↗
Fluorescent labelling of proteins on thin layers of solid dansyl chloride.
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Chemical Phenomena↗
Lipid mixing during freeze-thawing of liposomal membranes as monitored by fluorescence energy transfer.
A new pair of fluorescence-energy-transferring probes, dansylphosphatidylethanolamine and dioctadecylindocarbocyanine, were incorporated separately into phospholipid vesicles to monitor intervesicle lipid mixing under various conditions. The transfer efficiencies of mixtures of sonicated vesicles labeled with 2 wt% donor dansylphosphatidylethanolamine (DnsPE) or with 1 wt% acceptor dioctadecylindocarbocyanine (DiI-C18) were negligible, but increased to about 25% after the vesicles had been frozen in a solid CO2/ethanol bath, thawed and diluted. The freeze-thaw-induced mixing of lipids between vesicles, signified by energy transfer, was dependent on lipid concentration and was promoted by 0.5-1.5 M KCl, 0.5 M potassium trichloroacetate and 5 mM sodium acetate (pH 4) and inhibited by 0.5 M LiCl, 0.5 M glycerol, 0.5 M sucrose, 0.15 M KCl and 0.15-1.5 M NaCl. These results support and complement previously reported measurements of the trapped volumes, turbidities and population size distributions of similarly treated liposomes. Comparison of the responses of paucilamellar vesicles with those of multilamellar vesicles suggests that lipid mixing during freeze-thawing can occur either during interaction of the outermost bilayers of vesicles or during interaction of all bilayers, possibly as a result of breakdown and reformation of bilayer structure.
Dansyl Compounds↗
Enhancement of divalent anion transport across the human red blood cell membrane by the water-soluble dansyl chloride derivative 2-(N-piperidine)ethylamine-1-naphthyl-5-sulfonylchloride (PENS-Cl).
Sulfate transport across the red cell membrane is enhanced by the newly synthesised, water-soluble and nonpenetrating dansyl chloride derivative 2-(N-piperidine)ethylamine-1-naphthyl-5-sulfonylchloride (PENS-Cl). The transport is only enhanced if the potentiating agent 2-(4-aminophenyl-3-sulfonic acid)-6-methylbenzothiazol-7-sulfonic acid (APMB) is present during incubation with PENS-Cl. The enhanced flux is reduced by the anion-transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS) to about the same low level as in untreated controls. In contrast to dansyl chloride, PENS-Cl does not increase cation leakage from the red cells. The effects of PENS-Cl on sulfate transport resemble those produced by dansyl chloride. However, it can be shown that PENS-Cl only reacts with one subset of sites that are modified by dansyl chloride and involved in bringing about the enhancement of sulfate transport. This subset does not include the sites accessible to dansyl chloride in the absence of APMB. It comprises only a fraction of the sites exposed to dansyl chloride in the presence of APMB. Very little labelling of proteins of the red cell membrane can be seen after exposure of ghosts to the PENS-Cl, while dansyl chloride labels all major proteins.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
pH dependence of phosphate transport across the red blood cell membrane after modification by dansyl chloride.
Dansylation of the red blood cell membrane inhibits monovalent anion transport as measured by means of 36C1 and enhances divalent anion transport as measured by means of 35SO4 (Legrum, Fasold and Passow (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1573-1590 and Lepke and Passow (1982) J. Physiol. (London) 328, 27-48). In the present work the effect of dansylation on phosphate equilibrium exchange was studied over the pH range where the ratio between monovalent and divalent phosphate anions varies. At high pH, phosphate equilibrium exchange was enhanced; at low pH, exchange was inhibited. The pH maximum of phosphate equilibrium exchange, seen at pH 6.3 in untreated ghosts is now replaced by a plateau. The inverse effects of dansylation on the rates of exchange at high and low pH suggest that both monovalent and divalent phosphate anions are accepted as substrates by the anion transport protein. A tentative attempt to obtain a quantitative estimate of the ratio of monovalent and divalent phosphate transport indicates that in the untreated red cell membrane over the pH range 7.2-8.5 the transport of HPO42- is negligible compared to the transport of H2PO4-.
Anion Exchange Protein 1, Erythrocyte↗
Solvent relaxation in lipid bilayers with dansyl probes.
The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.
Dansyl Compounds↗
Fluorescent labeling of bacteriorhodopsin: implications for helix connections.
Purple membrane from Halobacterium halobium was reacted with dansyl (5-dimethylamino-1-naphthalenyl fluorescent labels that have specificity for different protein side chains of bacteriorhodopsin. Dansyl chloride was found to react primarily with Lys-41. Dansyl hydrazine was coupled, with water-soluble carbodiimide, to Glu-74 and/or Asp-85, which was the major modified site after papain-cleavage of the carboxyl-terminal 17 amino acids. Fluorescence energy transfer was used to probe the proximity of the modified sites to the retinal chromophore of bacteriorhodopsin. The dansyl group on Lys-41 was greater than 2.99 nm from retinal, while the dansyl group on Glu-74/Asp-85 was greater than 2.10 nm from retinal. Information available on the location of retinal in the transmembrane profile and probable surface locations of the fluorescent labels was combined with the energy transfer results to calculate distances projected in the plane of the membrane. The projected distances to retinal were 1.64 nm (Lys-41) and 1.65 nm (Gly-74). These measurements, combined with many other labeling experiments that have been reported, restrict the number of likely helix-connection models to only three: EDCABGF, FEDCBAG and FGEABDC (in the nomenclature of Engelman et al. (1980) Proc. Natl. Acad. Sci. USA 77, 2023-2027).
Bacteriorhodopsins↗
Effects of platelet activating factor on calcium-lipid interactions and lateral phase separations in phospholipid vesicles.
Recent studies localizing the inflammatory mediator, platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), to the membranes of stimulated neutrophils, raise the possibility that PAF may, in addition to its activities as a mediator, alter the physical properties of membranes. This, and the increasing evidence that calcium-lipid interactions may have central importance in membrane organizational structure and in functions of cell homeostasis and stimulus-response coupling, prompted us to study the effects of PAF on calcium-lipid interactions in lipid vesicles. Using fluorescence polarization of dansylated probes located in the glycerol portion of the membrane bilayer, PAF (at a concentration as low as 1 mol%) was shown to reduce membrane rigidification significantly during calcium-induced lateral phase separations. This effect of PAF was structurally dependent on both the 1-position alkyl linkage and the 2-position acetyl group as shown by studies of related lipid analogs. Furthermore, using a self-quenching probe, it was shown that inhibition of lateral phase separation did not account for this reduction in the calcium-induced membrane rigidification attributed to PAF. Data suggest that PAF at low concentrations may alter phospholipid head packing and, thereby, change membrane surface features during calcium-lipid interactions, effects which may ultimately explain some of its biological actions.
Animals↗
The effects of dansylation on the pH dependence of SO4(2-) and Cl- equilibrium exchange and on the H+/SO4(2-) cotransport across the red blood cell membrane.
Treatment of the erythrocyte membrane with dansyl chloride leads to the following effects: (i) SO4(2-) transport is enhanced, Cl- transport is reduced. At maximal acceleration of sulfate exchange, Cl- exchange is only partially inhibited. The two effects are lineary related suggesting that the Cl- and SO4(2-) transporting forms of band 3 are derived from the same pool. (ii) The maximum of the pH dependence of SO4(2-) equilibrium exchange as measured at low sulfate concentrations is replaced by a plateau. It now resembles the pH dependence of Cl- exchange in untreated red cells. The pH dependence of SO4(2-) equilibrium exchange as measured at high sulfate concentrations is virtually unchanged after dansylation. The pH dependence of the partially inhibited Cl- equilibrium exchange across the dansylated membrane as measured at high chloride concentrations remains similar as in the untreated red cells but is somewhat less pronounced. (iii) SO4(2-)/H+ cotransport remains essentially unaltered after modification by dansyl chloride. The effects of dansylation are discussed in terms of a model similar to the titratable carrier model originally proposed by Gunn (Gunn, R.B. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Rorth, M. and Astrup, P., eds.), pp. 823-827, Munksgaard, Copenhagen).
Biological Transport↗
Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome-b5 reductase systems in phosphatidylcholine vesicles.
The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeled cytochrome b5 (DNS-cytochrome b5), on interaction with NADH-cytochrome-b5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15-30 degrees C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15-30 degrees C. The enzyme activity of the DNS-cytochrome-b5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b5 and NADH-cytochrome-b5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.
Animals↗