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J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 397 records · Page 22Linked to original sources
PubMed1976

Binding of phosphorylcholine to an IgM Waldenström as studied by fluorescence spectroscopy and circular dichroism.

The binding characteristics of an IgM Waldenström(FR) for the ligand phosphorylcholine has been studied by fluorescence spectroscopy. Upon phosphorylcholine addition, IgM FR exhibited 83% enhancement of the tryptophanyl fluorescence, which was associated with a red shift of the emission maximun (5nm). The same properties were observed with the 7S IgM subunits. The association constant KA for phosphorylcholine was 6X10(4) M-1 FOR IgM FR and the 7S subunit, as determined by fluorescence titration, a value in agreement with the obtained by equilibrium dialysis. No significant decrease in the KA value was found in the presence of 3 M urea; in 6 M urea, the increase in fluorescence intensity was 36% of the value obtained in the absence of denaturing agent. In contrast, only 4% of fluorescence enhancement was noted upon binding in 3 M GuHC1 and no enhancement could be seen when the concentration of GuHC1 was increased to 5 M, thus suggesting complete unfolding of the protein and subsequent loss of binding activity. The pH dependence study of the phosphorylcholine binding to IgM FR indicated no significant differences in the fluorescence enhancement between pH 5 and 8, whereas at more acidic or alkaline pH values, the enhancement became smaller. At pH 3.0 and 10.0, no enhancement was seen suggesting no binding of the ligand, a fact confirmed independently by equilibrium dialysis. When the spectroscopic properties of the IgM FR were compared with those of murine myeloma proteins that bind the same ligand large differences were recorded in the amplitude of the phosphorylcholine induced enhancement of the fluorescnece and in the shift of the emission maximum wavelength. This suggests that the human and murine proteins interact differently with the small ligand phosphorylcholine thus implying that the variable domains of these molecules are not identical

Animals↗
PubMed1976

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.

Biological Transport↗
PubMed1976

Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.

Biological Transport↗
PubMed1976

Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

Age Factors↗
PubMed1976

Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts.

We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate.

Azides↗
PubMed1976

Measurement of lateral transport on cell surfaces.

This paper describes the principles and recent applications of two new methods for measuring rates of macroscopic lateral transport of fluorescent-labeled particles on the surface of individual cells. Both methods are based on microfluorimetric measurements of fluorescence intensity from a small open region (greater than or equal to 1 micronm radius) on the cell surface. Transport rates are measured from the rates at which the measured fluorescence intensity changes due to entrance or departure of fluorophores from the region. One method, "Fluorescence Photobleach Recovery" (FPR), uses a brief intense pulse of light to create an initial concentration gradient over the spot by irreversible photochemical destruction of fluorophores. The rate of fluorescence recovery due to transport of unbleached fluorophores into the observation region is the primary experimental datum. The other method, "Fluorescence Correlation Spectroscopy" (FCS), is based on a statistical analysis of spontaneous fluctuations of numbers of fluorophores in the observation region and does not require a perturbation to generate an initial concentration gradient. FCS is mainly useful to measure relatively fast processes (D greater than or equal to 10(-9) cm 2/sec) in stable systems; FPR can be used to measure both slow and fast transport in less stable systems. Using both FCS and FPR, the diffusion coefficient of a fluorescent lipid probe in rat myoblast plasma membranes was measured to be D = (9 +/- 4) X 10(-9) cm2/sec over a range of at least 4 micronm. FPR was used to measure the lateral mobility of the fluorescent labeled lectin concanavalin A complexed to myoblast plasma membrane "receptors." A fraction of the complexes were immobile on the time scale of the experiment (D less than 8 x 10(-12) cm2/sec). The remainder of the complexes had effective diffusion coefficients far smaller than expected from the measurements on the lipid probe (8 x 10(-12) less than or equal to D less than or equal to 3.3 x 10(-11) cm2/sec). Moreover the mobility depended on the valence, dose, and time of occupancy of the lectin on the membrane, suggesting that an aggregation of complexes was occurring during the experiment. Cytochalasin B decreased the mobility of complexes, suggesting an influence of microfilaments on the transport process. Neither azide nor colchine affected measured transport rates. These results indicate the operation of constraints on the mobility of the lectin-receptor complexes beyond that exerted by the viscous resistance of the lipid bilayer membrane matrix. The two types of interactions revealed by our current experiments - interactions of the complexes with microfilaments and with each other (aggregation) - seem insufficient to account entirely for the low observed mobility.

Animals↗
PubMed1976

The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size.

Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.

Animals↗
PubMed1975

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.

Antibodies, Bacterial↗
PubMed1975

Circular polarization of fluorescence of chlorophyll in solution and in native structures.

Chlorophyll dimers in solution, subchlorplast particles and chloroplasts were investigated by their circular dichroism and circular polarization of their fluorescence, which reflect their optical rotatory power in the ground state and electronically excited state, respectively. The chlorophyll dimers in fluid solution lose their optical activity upon electronic excitation, reflecting a marked concomitant change in the structure of the dimers. This change is arrested in a solution of very high viscosity. The pronounced difference between the circular polarization of the dimers in fluid media and that of subchloroplast particles and chloroplasts indicates that the former are not suitable models for associated chlorophyll in native structures in electronically excited states. Impairment of the photochemical activity of chloroplasts by heat treatment is accompanied by a reduction of the circular polarization of the fluorescence, which probably reflects a disorganization in structure. The same extent of circular polarization was observed in the fluorescence of chloroplasts regardless whether the reaction centers are open or closed; thus either the same molecules are emitting in the two cases or, if different molecules emit, they are packed in a similar way.

Binding Sites↗
PubMed1975

Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.

Amides↗
PubMed1975

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.

Circular Dichroism↗
PubMed1975

Antigen-induced conformational changes in antibodies and their Fab fragments studied by circular polarization of fluorescence.

Conformational changes induced in antibody molecules and in their Fab fragments by binding of antigen were investigated by the circular polarization of the fluorescence emitted by the tryptophan residues. This property of the fluorescence is related to the asymmetry, and thus to the conformation and environment, of the emitting chromophore. Changes in the circular polarization of the fluorescence of the antibody were observed upon binding of RNase to anti-RNase, of poly(DL-alanyl)-poly(L-lysine) to antipoly(D-alanine), and of the "loop" of lysozyme, a monovalent antigenic determinant, to anti"loop." The spectral changes were observed at different antigen-antibody ratios, including high antigen excess, indicating that they are due to antigen binding and not to aggregation. The circular polarization of fluorescence also detects changes in conformation of the different Fab fragments upon binding of the corresponding antigens. These changes in conformation were, however, markedly different from those observed for the whole antibody molecules, and indicated an interaction between the Fc and Fab fragments in the antibody molecule, and probably a change in the conformation of Fc upon binding of antigen to the antibody. In contrast, the small hapten, phosphorylcholine, did not induce a change in the circular polarization of the fluorescence of its antibody or corresponding Fab fragments. Reduction of the interchain disulfide bonds of the antibodies abolished the antigen-induced spectral changes due to the presence of the Fc portion in the molecule, but not the changes observed in Fab, suggesting that the disulfide bonds at the hinge region of the antibody are required for the transmission of the conformational change from the Fab to the Fc.

Animals↗