Physical properties of the lipid phase of membranes from cultured animal cells

Abstract

ESR analysis of membranes from cultured animal cells reveals a more complex lipid phase behavior than that displayed by ideal binary lipid systems. When endoplasmic reticulum membranes from LM cells are spin labeled with a nitroxide derivative of decane, 5N10, and scanned by ESR at 1°C-intervals, the partitioning of 5N10 between the hydrocarbon and aqueous portions of the memb5ane suspension undergoes thermotropic changes at characteristic temperatures of 9 , 16", 22", 32", and 38°C. Lipids extracted from these same membranes, however, exhibit only two characteristic temperatures, 16" and 35"C, and in this respect resemble binary lipid systems. The phase behavior of lipids in animal cell membranes is suggestive of an organized distribution of lipid which is disrupted by extraction. In support of this, mathematical treatment of the partitioning data indicates that four of these characteristic temperatures can define the boundaries (i.e., the t l and t, ) of two independent phase transitions in endoplasmic reticulum membranes. These results are similar t o those of a physical treatment of data from plasma membranes of both mouse and chick cells in which the two monolayers appear t o exist as independent physical entities with different physical properties. The most probable phase boundaries for the two monolayers of the endoplasmic reticulum membranes studied here are 16" and 32°C for one monolayer and 22" and 38°C for the other. INTRODUCTIONInvestigations of membrane lipid phase behavior have typically concentrated on model lipid systems and lipid-requiring strains of microbes. Physical studies on Escherichia coli membranes show that they behave like ideal binary lipid systems in that they display a single lateral phase separation process defined by lower and upper characteristic temperatures, t, and t,, respectively. These mark the beginning and end of melting of lipids in the membrane bilayer. Correlations between these physical characteristic temperatures and characteristic temperatures for membrane processes have been demonstrated in E. coli (1)(2)(3)(4)(5)(6)(7)(8) Recent studies by Wisnieski et al. (10) were undertaken to determine the physical state of the plasma membrane of LM cells, a cultured line of mouse fibroblasts. Electron spin resonance (ESR) studies revealed that extracted lipids from these membranes behaved like ideal binary lipid systems in that they displayed only two characteristic temperatures, a t, and a t h , Intact membranes, however, displayed four or more characteristic temperatures by ESR and by biochemical assays of amino acid transport and membrane ATPase. The unusual phase behavior of these membranes was shown toabe compatible with a membrane model in which the two monolayers of the membrane bilayer of animal cells exist as independent entities with different physical properties.In this report, we will summarize the physical and physiological evidence for four characteristic temperatures in the plasma membrane of LM cells and present the results of an ESR study of the physical stat...

Physical and Physiological Evidence for Two Phase Transitions in Cytoplasmic Membranes of Animal Cells

“…Keirns On the basis of the best agreement with the aggregate of physical and physiological data, we favor model 1, in which the characteristic temperatures are 220 and 380 for the inner monolayer and 14°and 32°for the outer monolayer of the membranous envelope of NDV. Similar characteristic temperatures (420) can be assigned to the inner and outer monolayers of the LM cell surface membrane, and this very same model best fits the ESR data for LM cell endoplasmic reticulum (21). This physical asymmetry has a compositional precedent in human erythrocyte membranes, where the bulk of the choline containing phosphatides is apparently localized on the outer monolayer, and phosphatidylserine and phosphatidylethanolamine are on the inner monolayer (35,36).…”

“…Cytoplasmic membranes were isolated from mouse LM cells maintained as monolayer cultures in Eagle's minimal essential medium (MEM) l)lus 0.5%lpel)tone (19). Membranes were isolated as described by Schimmel et al (20) with minor modification (21 Bligh and Dyer (23). The lipid extract (0.4 ml) was filtered through Na2SO4 and glass wool, dried under a stream of nitrogen gas, and suspended in 80 Al of chloroform: methanol (2: 1).…”

Fluorescence polarization studies of rat intestinal microvillus membranes.

“…In addition to fluorescent probe analysis the techniques used include differential scanning calorimetry (44), X-ray diffraction (45), electron spin resonance (46)(47)(48), niuclear magnetic resonance (49,50), and the temperature dependence of various enzyme and transport functions (43,51). Evidence for phase transitions in mammalian membranes, however, has been limited to mouse LH cells cultured in monolayers (52,53) and studied by electron spin resonance and the nitroxide spin label, SN10, in association with a numiber of physiological parameters. The present study provides the first clear demonstration of a phase transition in plasma membranes isolated directly from a normal mammalian tissue.…”

“…The present study provides the first clear demonstration of a phase transition in plasma membranes isolated directly from a normal mammalian tissue. While the mean transition temperature of approximately 26°C is sufficiently below 37°C to preclude a physiological role for the change in state, the transition phenomenon should prove useful in further studies of membrane transport and enzyme reactions as it has in other cell types (43,52,53).…”

Differential lateral mobility of IgM and IgG receptors in mouse B lymphocyte membranes

“…Recent evidence suggests that the asymmetric distribution of lipids and proteins across the bilayer membrane of animal cells may result in a physically asymmetric bilayer (8). In accordance with this model, it has been shown that the inner and outer monolayers of the membrane bilayer display two nonidentical sets of characteristic temperatures (9)(10)(11).…”