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R D Klausner

Publications and source records attributed to R D Klausner.

At least 253 records · Page 14Linked to original sources
PubMed1982

Compartmental analysis of light-induced proton movement in reconstituted bacteriorhodopsin vesicles.

Purified bacteriorhodopsin from purple membrane sheets isolated from Halobacter halobium was solubilized with a bile salt detergent, 3-[(3-cholamidopropyl) dimethyl-ammonium]-1-propanesulfonate (CHAPS). The detergent-solubilized protein was then incorporated into lecithin vesicles at either high (450:1) or low (65:1) lipid to protein ratios. Circular dichroism studies showed that the bacteriorhodopsin incorporated was in a monomeric form in the 450:1 vesicles. The 65:1 vesicles exhibited an exciton splitting characteristic of the aggregated state of bacteriorhodopsin. We then examined the light-induced movement of protons for these two preparations. Compartmental analysis was used to derive a kinetic model for the observed proton movement. The pumping was qualitatively the same for monomeric and aggregated protein. A three-compartment model provided an excellent description of proton movement in both sets of vesicles and at four different light intensities. This model demands two independent processes to account for the proton movement. The rate coefficients for both are linearly related to light intensity. However, the total flux of protons via one of these processes diminishes as a function of the hydrogen ion accumulation within the vesicles.

Bacteriorhodopsins↗
PubMed1982

Prediction of the three-dimensional structure of the leader sequence of pre-kappa light chain, a hexadecapeptide.

The three-dimensional structure of the signal sequence for murine kappa light chain has been calculated by using conformational energy calculations. These calculations, based on tested and reliable potential energy functions, employ a novel global search technique to identify the lowest energy structures for the hexadecapeptide signal sequence, Glu-Thr-Asp-Thr-(Leu3-Trp-Val)2-Pro-Gly.l It has been found that the core hydrophobic sequence, Leu3-Trp-Val-Leu3, adopts an alpha-helical conformation that is terminated by chain reversal conformations for the four residues, Trp-Val-Pro-Gly. The amino-terminal four residues adopt low energy conformations that are fully compatible with the succeeding alpha-helix. The immediately neighboring sequence., Asp-Thr, exists in a single lowest energy double-equatorial conformation, whereas the first two residues, Glu-Thr, can adopt a variety of low energy conformations. The calculations arrive at a highly structured and specific model for the conformation of a leader sequence, compatible with recent experimental data.

Amino Acid Sequence↗
PubMed1982

Calculation of the three-dimensional structure of the membrane-bound portion of melittin from its amino acid sequence.

The structure of the NH2-terminal, 20-residue membrane-bound portion of melittin has been computed with empirical energies (ECEPP, Empirical Conformational Energy Program for Peptides). First, a search was made for long stretches of nonpolar residues. Then, the low-energy conformations of these segments were built up by combining successively the low-energy conformations of their component di- and tripeptides. The minimum-energy conformations of each of these component peptides used in this buildup process were selected so that each had a distinct backbone conformation; these distinct backbone conformations were designated as nondegenerate minima. Structured segments (i.e., those with only a few low-energy conformations) resulting from this process were identified and used as nucleation sites for building up larger structures by adding adjacent peptide segments. At each stage, the energy of each structure was minimized. Only two low-energy structures were found, both of which were alpha-helical from Gly-1 to Thr-10 and from Pro-14 to Ile-20. In one of them, the first helix continues through Thr-11-Gly-12; in the other, there is a bend at Thr-11-Gly-12. However, because of compensating changes in the dihedral angles, both structures are very similar. The calculated structures seem to agree well with experimental data.

Amino Acid Sequence↗
PubMed1982

Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes.

The endocytosis leading to degradation of 125I-labeled asialo-orosomucoid specifically bound to the surface of freshly isolated hepatocytes was monitored as a function of time at 37 degrees C. Experimental values were determined for the rates of internalization, dissociation of the receptor-ligand complex, and degradation of the labeled ligand. Compartmental analysis and computer modeling revealed that the data were compatible with dissociation of ligand from receptor preceding ligand degradation. The rate coefficient for internalization was calculated to be an order of magnitude greater than that for receptor--ligand dissociation. Ligand internalization did not result in concomitant depletion in the total number of cell surface receptors. Our data are taken to indicate that ligand remains associated with the receptor after internalization, that the complex is dissociated prior to degradation, and that new, unoccupied receptors are promptly returned to the cell surface from an internal pool.

Animals↗
PubMed1982

Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment.

At physiological temperature, the Fe-carrier transferrin is taken up by K562 human erythroleukemia cells through receptor-mediated endocytosis. Both ligand (now minus Fe) and receptor recycle back to the cell surface where the receptor is rapidly reutilized. After endocytosis, transferrin becomes transiently lodged within an acidic compartment inside the cell, as judged by the changed spectral characteristics and quantum yield of fluorescein isothiocyanate-labeled transferrin that is cell-associated at 37 degrees C. Upon binding to transferrin, anti-fluorescein antibody strongly quenches the emission of the fluorescein-labeled residues on the protein and is used to assess whether the transferrin is at the cell surface (incubation at 0 degrees C) or mainly internalized into the cell (incubation at 37 degrees C). Using Percoll gradient fractionation of postnuclear supernatants, we show that the acidic compartment is not the lysosomal compartment.

Cell Compartmentation↗
PubMed1981

The lack of relationship between fluorescence polarization and lateral diffusion in biological membranes.

An investigation has been carried out of the relationship between changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and concomitant changes in the lateral diffusion of proteins and lipid probes in membranes. Plasma membranes from lymphocytes and a CH1 mouse lymphoma line were treated with up to 70 mol% (relative to the total membrane phospholipid) of oleic or linoleic fatty acids. Under these conditions the fluorescence polarization of DPH decreased by between 8 and 15% which, in the framework of the microviscosity approach, suggests a membrane fluidity change of between 20 and 50%. The lateral diffusion coefficients of surface immunoglobin and the lipid probes 3,3'-dioctadecylindocarbocyanine and pyrene were also measured in these membranes using the fluorescence photobleaching recovery technique and the rate of pyrene excimer formation. The diffusion rates were found to be unaffected by the presence of free fatty acids. Hence despite large 'microviscosity' changes as reported by depolarization of DPH fluorescence, lateral diffusion coefficients are essentially unchanged. This finding is consistent with the idea that perturbing agents such as free fatty acids do not cause a general fluidization of the membrane but act locally to alter, for example, protein function. It is also consistent with the suggestion that lateral mobility of membrane proteins is not modulated by the lipid viscosity.

Animals↗
PubMed1981

Interaction of tubulin with phospholipid vesicles. I. Association with vesicles at the phase transition.

Purified soluble tubulin will interact with highly sonicated small unilamellar vesicles of dipalmitoyl phosphatidylcholine. This results in the leakage of a fluorescent dye, carboxyfluorescein, from the internal aqueous space of the vesicles at the lipid phase transition. The amount of dye leaked from the vesicles is determined by the tubulin to vesicle ratio and partial dye leakage reflects the uniform leakage of carboxyfluorescein from all of the vesicles. Once tubulin interacts with dipalmitoyl phosphatidylcholine vesicles at the phase transition, it is unavailable to interact with additional vesicles. This interaction represents the formation of stable tubulin-vesicle recombinants as shown by density gradient centrifugation. Such recombinants are only formed by interacting the protein with the vesicles at the lipid phase transition temperature. The interaction is insensitive to ionic strength (0.001 to 4 M NaCl) and the recombinants remain stable for days.

Animals↗
PubMed1981

Interaction of tubulin with phospholipid vesicles. II. Physical changes of the protein.

We have shown that soluble tubulin will bind to small unilamellar vesicles of dipalmitoyl phosphatidylcholine (Klausner, R. D., Kumar, N., Weinstein, J. N., Blumenthal, R., and Flavin, M. (1981) J. Biol. Chem. 256, 5879-5885). This association uniquely occurs at the lipid phase transition. The tubulin, when bound to the vesicles, displays an altered tryptophan fluorescence characterized by a 5-nm blue shift in the emission maximum and a 22% decrease in fluorescence intensity, when compared to soluble tubulin. Tryptophans in vesicle-bound tubulin are less accessible to the aqueous collisional quenchers, acrylamide and iodide, than in soluble tubulin. Circular dichroism studies reveal an increase in alpha-helical content of tubulin as a result of vesicle interaction. Proteolytic digestion by trypsin of vesicle bound tubulin is slower than of soluble tubulin. The beta subunit of tubulin is preferentially protected from trypsin by vesicle interaction. Furthermore, the pattern of tryptic cleavage products is altered by this interaction.

Acrylamides↗
PubMed1981

Effects of free fatty acids on the organization of cytoskeletal elements in lymphocytes.

Treatment of mouse lymphocytes with cis-unsaturated free fatty acids produced alterations in the immunofluorescence patterns of the cytoskeleton and contractile proteins. Saturated free fatty acids and trans-unsaturated free fatty acids had no effect. In untreated cells, the microtubular pattern exhibited radiation from an organizing center, resembling the spokes of an umbrella. The addition of linoleic acid produced a polarized submembranous aggregate. Under control conditions, staining for actin revealed a diffuse pattern over the entire cell, but the addition of linoleic acid caused the formation of a single large patch, or polarized submembranous aggregate. The pattern for alpha-actinin normally revealed intense perinuclear staining on a diffuse background. Linoleic acid caused the loss of this pattern and the formation of a polarized submembranous aggregate. Linoleic acid treatment also caused the pattern for myosin to change from diffuse to uniform submembranous patching around the periphery of the cell. For all of these proteins, calcium (8 mM), but not magnesium, partially reversed the effects of linoleic acid. Sodium azide had little effect on the normal distribution of actin, tubulin, and alpha-actinin; however, myosin staining revealed prominent patch formation. Colchicine treatment caused diffuse staining, some polarized submembranous aggregate formation of tubulin, and some patching of myosin, but not as extensively as did treatment with linoleic acid. Actin and alpha-actinin were unaffected. These results, in view of the previously shown facts that pretreatment of cells with linoleic acid followed by anti-immunoglobulin inhibits capping of surface immunoglobulin (Klausner, et al., Proc. Natl. Acad. Sci. U.S.A. 77:437-441, 1980) and that free fatty acids partition into the surface membrane (Klausner et al., J. Biol. Chem. 255:1286-1295, 1980), suggest that the perturbation of the plasma membrane with unsaturated free fatty acids alters the interaction of surface receptors with the cytoskeleton, which in turn affects cytoplasmic distribution of the proteins.

Actins↗
PubMed1980

Selectivity of fluorescent lipid analogues for lipid domains.

We have examined the phase partition preferences of the even chain length (n = 10-22) diacyl-3'3'-indocarbo-cyanine iodides (Cn diI) incorporated in disaturated lecithin (PC) vesicles. Two parameters were used to determine this phase preference: (i) the direction of shift of the phase transition temperature (Tm) induced by the dyes and (ii) the self-quenching of fluorescence due to aggregation in the gel phase of those dyes which preferentially partition into the fluid. Dyes that lower Tm preferentially partition into the fluid phase; those that raise Tm preferentially partition into the gel. By these criteria in dimyristoyl-PC, C10 diI and C12 diI preferentially partition into the fluid phase, C14 diI and C16 diI show no preferential partition, C18 diI preferentially partitions into the gel, and C20 diI and C22 diI preferentially partition into the fluid. In dipalmitoyl-PC, the pattern of preference is identical with that observed in dimyristoyl-PC, only shifted to longer chain length diI's by two carbons. Diffusion measurements by fluorescence photobleaching recovery of these dyes in gel-phase multilayers showed tham all to be immobile, D less than 10(-10) cm2/s, while in fluid-phase multilayers thay all had diffusion coefficients of D approximately 10(-8) cm2/s independent of chain length. In mixed-phase multilayers, however, each Cn diI showed mobile fraction which reflected its phase-partition preference.

Chemical Phenomena↗
PubMed1980

Voltage-dependent translocation of the asialoglycoprotein receptor across lipid membranes.

A membrane receptor protein for asialoglycoproteins induces voltage-dependent increases in ion conductance across a lipid bilayer, probably reflecting penetration of the protein into the bilayer towards an electrically positive pole. In the presence of specific ligand for the receptor, this penetration leads to a 'translocation' of the receptor from one side of the bilayer to the other. These observations suggest a mechanism by which biological membranes might regulate the disposition of their proteins, and a way in which membrane receptors involved in endocytosis might be spared lysosomal destruction in order to be recycled to the plasma membrane.

Animals↗
PubMed1980

Model for capping derived from inhibition of surface receptor capping by free fatty acids.

When low concentrations (2-5 mole %) of cis unsaturated free fatty acids (group A) are intercalated into lymphocyte plasma membrane, capping is inhibited. No effect is seen with trans unsaturated or saturated fatty acids (group B). The capping inhibition is reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery has shown that the group A free fatty acids do not inhibit the receptor immobilization associated with patch formation, but inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the group A free fatty acids cause a shift in membrane-bound calcium to the lipid phase from probable protein-associated sites. We have incorporated these findings into a model for capping and membrane-cytoskeletal interactions.

Animals↗