Immunochemistry of Model Membranes Containing Spin-Labeled Haptens

294

215

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 23.80C, the chain-melting transition temperature of this phosphatidylcholine. There is a temperature-composition region, approximately temperature less than 230C 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 z230C there is a significant increasing cholesterol concentration, for X > 0.2.The physical properties of bilayer membranes containing cholesterol and phosphatidylcholines have been the subject of extensive investigations employing a large number of experimental techniques (1-16). From these studies certain generalizations emerge. The addition of cholesterol to phosphatidylcholine bilayers in the fluid state (above the chain-melting temperatures of the phosphatidylcholines-i.e., above 23.80C for dimyristoyl phosphatidylcholine), leads to a decrease in the "fluidity" of the bilayer membrane. On the other hand, inclusion of cholesterol in phosphatidylcholine membranes at temperatures below the chain-melting transition temperatures leads to "fluidization" of these membranes (1-16). These generalizations have been based in part on magnetic resonance studies (spin-label paramagnetic resonance and nuclear magnetic resonance) in which the spectra are sensitive to the rotational motions of the molecules. Another interesting property of binary mixtures of dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine with cholesterol is that a phosphatidylcholinelike phase transition (at t230C or -420C) can be detected in the presence of mole fraction X < 0.20 cholesterol in the binary mixture (1,3,6,7,9,10,12,16). The chain-melting transition temperature of dimyristoyl phosphatidylcholine is 23.80C, and that of dipalmitoyl phosphatidylcholine is 41.40C. Although reliable phase diagrams have been established for a number of binary mixtures of phospholipids (17-20), it has been difficult to use the published phase diagrams for binary mixtures of cholesterol and phosphatidylcholines (3, 4, 7) to account for all of the observed physical properties of these mixtures. Some of this difficulty may originate in sample heterogeneity (4).In the present paper we give the results of determinations of the lateral diffusion coefficient of the fluorescent probe phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazole)ethanolamine (structure I; derived from egg phosphatidylcholine) in binarymixtures of cholesterol and dimyristoyl phosphatidyicholine as a function of temperature and composition. Some diffusion coefficients for this probe in binary mixtures of dipalmitoyl phosphatidylcholine and cholesterol are also given. As will be seen, such data shed considerable light on the nature...

mentioning

confidence: 99%

Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines.

Rubenstein¹,

Smith²,

McConnell³

1979

294

215

“…Likewise, specific antierythrocyte antibodies of the IgM class are about 103 times more efficient in C-mediated lysis than are specific IgG antibodies (3,4). On the other hand, efficiencies of the same effector components against different target membranes can differ widely (5)(6)(7)(8)(9)(10). In attempting to elucidate mechanistic differences in molecular terms, we have turned to the use of model membranes (liposomes or vesicles) to serve as targets for these various components of the immune system (7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

Membrane-controlled depletion of complement activity by spin-label-specific IgM

Humphries

McConnell

1977

Complement depletion mediated by high molecular weight (IgM) rabbit antibodies specifically bound to spin-label lipid haptens dispersed in model membranes is controlled by various physical attributes of those membranes other than the total number of exposed determinants that they provide. Carrier lipids used at 320 were (i) a "fluid" phosphatidylcholine (PC), (if) a "solid" PC, and (iil) a cholesterol/PC mixture.The concentration of hapten in the plane of the membranes (two-dimensional concentration) was varied while the overall hapten molarity (three-dimensional concentration) was kept constant.Both specific binding and the efficiency of depletion by IgM are markedly enhanced by systematically decreasing the average distance between haptens (co -_ 26 A). Heterogeneous distribution was found to be more favorable than a random homogeneous distribution of the same number of haptens in the same total quantity of lipids. IgM efficiency is also markedly increased by the inclusion of cholesterol in PC membranes, an effect thought to result from enhanced projection of the determinant from the surface of the membrane and hence increased accessibility to the antibody-binding site. Furthermore, the efficiency of IgM was increased by using haptens dispersed in fluid rather than in solid PC membranes.The results are consistent with the hypothesis that IgM molecules must be bound to a critical multiple of antigenic determinants at a membrane surface in order to induce complement-mediated attack and that subtle variation of the physical state of membrane antigens can be the crucial factor in determining the outcome of this type of efferent immune response.The relative cytotoxic efficiencies of different effector components of the immune system against the same target membrane can differ by several orders of magnitude. For example, erythrocyte cytolysis by antibody-dependent monocytes is typically about 103 times more efficient per IgG antibody molecule than is cytolysis of the same cells with the same antibody in the presence of complement (C) (1, 2). Likewise, specific antierythrocyte antibodies of the IgM class are about 103 times more efficient in C-mediated lysis than are specific IgG antibodies (3, 4). On the other hand, efficiencies of the same effector components against different target membranes can differ widely (5-10). In attempting to elucidate mechanistic differences in molecular terms, we have turned to the use of model membranes (liposomes or vesicles) to serve as targets for these various components of the immune system (7-12). Because the physical and chemical properties of such target membranes can be varied, we can hope to discern which special characteristics of an immune response are directly related to properties of components of the immune system and whichThe costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in...

“…Perhaps the most interesting question is the extent to which such motions and distributions are involved in cell surface recognition. With these broad problems in mind, we have undertaken a study of the immunochemistry of model membranes (e.g., liposomes) in which the structure, motion, distribution, and concentration of membrane haptens can be established by physical and chemical methods (7,8). Our studies take advantage of the discovery by Kinsky and colleagues that hapten-sensitized liposomes can be damaged by specific antibodies Abbreviations: DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; iodoacetamide spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; Tempo, 2,2,6,6-tetramethylpiperidine-l-oxyl; PBS, 10 mM sodium phosphate-buffered saline, pH 7.3,150 mM NaCl; Tempo-choline chloride, N,N-dimethyl-N-(2',2',6',6'-tetramethyl-4'-piperidinyl-1'-oxyl)-2-hydroxyethylammonium chloride.…”

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confidence: 99%

“…New Zealand rabbits were immunized as described before (7,8). The sera obtained were assayed qualitatively by 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) binding (see below).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Lateral hapten mobility and immunochemistry of model membranes.

Brûlet¹,

McConnell²

1976

A study has been made of the interaction of specific anti-nitroxide (anti-spin label) lgG antibodies, Fab fragments, complement, and liposomes containing dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine plus 0-50 mole % cholesterol, together with various concentrations of a head group spin-labeled phospholipid (0.5-0.01 mole % in the plane of the membrane). The spin-labeled lipid is the amide formed from the reaction of an iodoacetamide spin label, N(1-oxyl-2,2,6,6-tetramethyl4fpiperidinyliodoacetamide, with dipalmitoylphosphatidylethanolamine. We have reached two conclusions: (i) complement fixation depends on the lateral mobility of the lipid hapten at low hapten concentrations in the plane of the membrane (.<0.1 mole %) and does not depend strongly on this mobility at high hapten concentrations; (ii) cholesterol may have two distinct effects on complement fixation, one arising from an enhancement of hapten exposure to antibody binding sites and a second due to a modulation of membrane fluidity. There is much evidence to suggest that the lateral motions and distributions of membrane components may play significant and complement (9, 10). The present work provides strong evidence that the fixation of complement by liposomes containing lipid hapten spin labels and specific antibodies to spin labels depends on the lateral mobility of the hapten when the hapten concentration in the plane of the membrane is low. The present work also clarifies and extends our previous report (11) of the enhancement of complement fixation by the inclusion of cholesterol in liposomal membranes. The inclusion of cholesterol enhances antibody binding, probably by increasing hapten exposure at the membrane surface, in addition to modulating membrane fluidity. MATERIALS AND METHODSSpin-Label Lipid Hapten. The spin-label lipid hapten I was the reaction product of an iodoacetamide spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide, and L-a-dipalmitoylphosphatidylethanolamine. H2C-O-CO-(CH2)14-CH3