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Biomedical subjects

H M McConnell

Publications and source records attributed to H M McConnell.

At least 19 recordsLinked to original sources

Lateral diffusion of M-13 coat protein in model membranes.

A fluorescent derivative of the M-13 phage coat protein (molecular weight 5260) was reconsituted into oriented multilayers and giant liposomes of dimyristoylphosphatidylcholine. The rate of lateral diffusion of the labeled protein in the fluid phase was measured as a function of temperature and found to be comparable to that of lipid probes. The protein was found to have a nonuniform lateral distribution in the solid phase of both types of model membranes. Cardiolipin (0.5 mol %) included in the multibilayers did not have any substantial effect upon the rate of diffusion.

Coliphages

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.

Cholesterol

Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines.

The lateral diffusion of a fluorescent-labeled phospholipid, phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3,-diazole)ethanolamine, has been measured in binary mixtures of cholesterol and dimyristoyl phosphatidylcholine at temperatures both above and below 23.8 degrees C, the chain-melting transition temperature of this phosphatidylcholine. There is a temperature-composition region, approximately temperature less than 23 degrees C and mole fraction of cholesterol (X) less than 0.20, in which the lateral diffusion coefficient of the fluorescent probe is at least an order of magnitude smaller than it is at points outside of this temperature-composition region. At temperatures above approximately 23 degrees C there is a significant increasing cholesterol concentration, for X greater than 0.2.

Cholesterol

Theory of protein-lipid and protein-protein interactions in bilayer membranes.

A model for protein-lipid interactions in bilayer membranes where the proteins are very dilute is extended to higher protein concentration, where appreciable lipid-mediated protein-protein interactions occur. It is found that proteins may change the lipid phase transition temperature and that they weaken the phase transition. There exists a critical protein concentration above which the sharp lipid phase transition is abolished. The model also qualitatively reproduces several experimental observations on the physical behavior of bilayers formed from mixtures of cholesterol and phosphatidylcholines.

Cholesterol

Anisotropic molecular motion on cell surfaces.

Photobleaching in a two-dimensional periodic pattern has been used to characterize the motion of succinylconcanavalin A receptors on the surfaces of mouse fibroblasts. These receptors were found to exhibit anisotropic two-dimensional diffusion in adherent cells having parallel stress fibers. Diffusion coefficients of the order of 10(-11) cm2/sec and 10(-12) cm2/sec were measured for diffusive motions parallel and perpendicular to these fibers. The determination of the anisotropic motion used a two-dimensional Fourier analysis. A study was made of the effect of changes in cell shape on the photobleach pattern.

Animals

Specificity of memory cells raised against trinitrophenyl-conjugated syngeneic cells.

Exogenous lipid analogues can be incorporated into purified plasma membranes of EL4 tumor cells. When EL4 membranes are incubated with sonicated lipid vesicles containing spin-labeled phosphatidylcholine and then purified, they exhibit paramagnetic resonance spectra characteristic of spin labels dilutely dispersed in the lipid bilayer. Also, when EL4 membranes are incubated with vesicles of fluorescent lipid (N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine), the fluorescence intensity of the mixture gradually increases and the labeled membranes are strongly fluorescent under a microscope, whereas the original lipid vesicles are much less fluorescent because of self-quenching at the high relative concentration of the fluorophore in the vesicles (33%, wt/wt). Thus, lipid analogues can be integrated into the bilayers of EL4 plasma membranes. The ability of the EL4 membranes containing trinitrophenyl phosphatidylethanolamine to induce a secondary proliferation of C57BL/6 mouse spleen lymphocytes primarily stimulated by trinitrophenyl-modified autologous cells was investigated. Although trinitrophenyl-modified plasma membranes as well as trinitrophenyl-modified lymphocytes or EL4 cells caused a specific, H-2 restricted response, membranes containing trinitrophenyl phosphatidylethanolamine did not induce a secondary proliferative response. Because of the strong likelihood that all three lipid analogues mentioned above are similar to one another in having a high lateral mobility and in not being strongly associated with H-2 cell-surface molecules, these results can be viewed, with caution, as being consistent with a "modified self" model rather than a dual recognition model for H-2 restriction in the cell-mediated immune response to chemically modified syngeneic cells.

Animals

Allogeneic cytolysis of reconstituted membrane vesicles.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

Animals

Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.

Antigen-Antibody Complex

Reconstitution of the erythrocyte anion channel.

Band 3, the membrane protein which mediates erythrocyte anion exchange, was purified on a concanavalin A column. Triglycerides, diglycerides, cholesteryl esters, cholesterol, and phosphatidylcholine were found to copurify. The column product gave at least two and probably three bands by isoelectric focusing. Antibodies prepared against the purified Band 3 appeared to react only with the cytoplasmic face of Band 3. Vesicles prepared with Band 3 had an accelerated uptake of SO4(2-) which could be inhibited by 2-(j'-aminophenyl)-6-methyl benzene thiazo-3', 7-disulfonic acid, a potent inhibitor of anion transport in the intact system. The possible source of this difference is discussed. Band 3 was spin labeled, probably at one specific site. The spectra showed that the spin label was highly immobilized, but no dipole-dipole interactions between spin labels on adjacent Band 3 subunits were apparent.

Anions

Multiple phase equilibria in binary mixtures of phospholipids.

Approximate phse diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidycholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data. These phase diagrams represent an improvement over previous studies in that both solid phses (Pbeta' and Lbeta') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two Pbeta' phases that do not form a continuous range of solid solutions.

Freeze Fracturing

Surface areas of lipid membranes.

Upon photolysis, alkyl pentacyanocobaltate complexes generate alkyl radicals which react rapidly and specifically with nitroxide radicals, and which do not penetrate phospholipid bilayers. By measuring the loss of paramagnetic resonance signal intensity when multilamellar liposomes containing a small amount of spin-labeled lipid are exposed to these radicals, we have measured the proportion of lipid on the external surface of liposomes. We have shown that liposomes prepared under specified conditions from dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and binary mixtures of dipalmitoylphosphatidylcholine and cholesterol all have the same proportion of external lipid.

Cholesterol

Specific antibody-dependent binding of complement component C1q to hapten-sensitized lipid vesicles.

The binding of a component of human complement (C1q) to membrane-bound specific anti-nitroxide antibodies was studied as a function of the physical properties (fluid vs. solid) of vesicle lipids. The antibodies were bound to spin-label lipid haptens in the vesicle membrane. The binding of C1q to the sensitized vesicles shows a maximum as a function of specific IgG concentration. The binding of antibodies and of C1q to the vesicle membrane does not depend strongly on the physical state of the membrane lipids. even at low hapten (0.05 mol%) concentrations. These results are of significance for the understanding of the previously reported effect of lipid physical states on complement depletion.

Antibodies

Theoretical study of protein--lipid interactions in bilayer membranes.

An analysis is given for the perturbation of the order and composition of lipid bilayers near an intrinsic membrane protein. Two cases are examined: the protein influences the lipid order (i.e., "fluidity"), and the protein associates with one component of a lipid mixture preferentially. The order perturbation is studied as a function of temperature and lateral pressure by using Landau--de Gennes theory and a variational procedure. It is concluded that, for a given lateral pressure, the greatest amount of boundary lipid is present at the lipid phase-transition temperature. A critical point for the phase transition occurs, near which the amount of boundary lipid increases dramatically. The composition perturbation is modeled in a binary lipid mixture by using a simple regular solution theory. The perturbation is found not to extent much beyond the directly bound layer of lipids unless the solution is near a critical mixing point.

Chemical Phenomena

Determination of molecular motion in membranes using periodic pattern photobleaching.

The lateral diffusion of a fluorescent phospholipid probe in oriented multibilayers of dimyristoylphosphatidylcholine has been measured by observing the redistribution of fluorescence after photobleaching of the membranes in a periodic pattern of parallel stripes. The diffusion constant D of the fluorescent lipid was found to vary between 1.5 X 10(-11) cm2/sec at 9.6 degrees and 2.0 X 10(-10) at 22.5 degrees in the monoclinic phase. Preliminary studies of dipalmitoylphosphatidylcholine liposomes in the Lbeta' and Pbeta' phases yielded diffusion constants of the order of 10(-11) cm2/sec. These data are relevant to earlier discussions of the rate of complement activation by hapten-sensitized liposomal membranes [Brûlet, P. and McConnell, H. M. (1976) Proc. Natl. Acad. Sci. USA 73,2977--2981; Parce, J. W., Henry, N. and McConnell, H.M. (1978) Proc. Natl. Acad. Sci. USA 75, 1515--1518)]. We have also used this method to study the motion of fluorescent antibodies bound to murine EL-4 tumor cells. Pattern photobleaching techniques have the advantages that cellular or liposomal translation has no major adverse effect on the measurements, that certain nondiffusive motions can be detected and characterized, and that diffusive or other motions can be recorded photographically.

Diffusion

Visualization of specific antibody and C1q binding to hapten-sensitized lipid vesicles.

Specific IgG antibodies directed against the spin-label nitroxide group present as a lipid hapten in single-compartment lipid vesicles have been visualized by using freeze-etch electron microscopy. Individual "particles" with diameters of the order of 20 nm are identified as single IgG molecules bound to lipid hapten. No significant aggregation of these IgG molecules was observed over a period of 1 hr in a dipalmitoyl phosphatidylcholine vesicle at 22 degrees. Binding of the human complement component C1q results in the formation of large (approximately 50-100 nm) asymmetric particles having a partially resolved substructure that may arise from individual IgG molecules bound to the membranes as well as to C1q. The binding of C1q appears to result in a clustering of membrane-bound IgG molecules. Samples containing a serum factor (perhaps anti-IgG antibodies) exhibit some IgG clustering distinct from that produced by Clq.

Complement C1