Lipid–Protein Interactions in Rat Renal Subcellular Membranes: A Biophysical and Biochemical Study

• Published in: Archives of biochemistry and biophysics 2000-10, Vol.382 (1), p.39-47
• Main Authors: D'Antuono, Cecilia, del Carmen Fernández-Tomé, María, Sterin-Speziale, Norma, Bernik, Delia L.
• Format: Article
• Language: English
• Subjects: 2-Naphthylamine - analogs & derivatives; 2-Naphthylamine - pharmacology; Animals; Cell Membrane - chemistry; Cell Nucleus - chemistry; Cholesterol - chemistry; Chromatography, Thin Layer; Endoplasmic Reticulum - chemistry; Fatty Acids - chemistry; Fluorescent Dyes - pharmacology; Intracellular Membranes - chemistry; Kidney Medulla - chemistry; Kidney Medulla - metabolism; Laurates - pharmacology; Laurdan generalized polarization; Lipid Bilayers - chemistry; Lipid Metabolism; Lipids - chemistry; Liposomes - chemistry; Male; phase behavior; Phospholipids - chemistry; Proteins - chemistry; Proteins - metabolism; Rats; Rats, Wistar; renal papilla membranes; Spectrometry, Fluorescence; Subcellular Fractions - chemistry; Temperature
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2000.1979

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5°C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20°C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin—the latter being a highly saturated phospholipid—and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.