PERFLUOROALKYLPHOSPHOCHOLINES ARE POOR PROTEIN-SOLUBILIZING SURFACTANTS, AS TESTED WITH NEUTROPHIL PLASMA-MEMBRANES

Citation
C. Dermardirossian et al., PERFLUOROALKYLPHOSPHOCHOLINES ARE POOR PROTEIN-SOLUBILIZING SURFACTANTS, AS TESTED WITH NEUTROPHIL PLASMA-MEMBRANES, Biochimie, 80(5-6), 1998, pp. 531-541
Citations number
62
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0300-9084
Volume
80
Issue
5-6
Year of publication
1998
Pages
531 - 541
Database
ISI
SICI code
0300-9084(1998)80:5-6<531:PAPPS>2.0.ZU;2-M
Abstract
We have tested the membrane-protein solubilizing properties of two per fluoroalkylphasphocholines. These compounds belong to a series of fluo rinated amphiphiles which are being investigated as potential stabiliz ing agents for a variety of fluorocarbon-based systems. We are particu larly interested in cytochrome b(558) from phagocytes, the redox compo nent of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This coul d be due to a delipidating, denaturing effect of these detergents (oct yl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F 17(CH2)(2)O-P(O-2(-))-O(CH2)(2)N+ (CH3)(3)(F8C2PC), extracted about as much protein from neutrophil plasma membranes into 100 000 g supemata nt as octyl glucoside. The second compound, C8F17(CH2)(11)O-P(O-2(-))- O(CH2)(2)N+(CH3)(3) (F8C11PC), was less efficient. We found that flavi n was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained with out addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside e xtracts. These experiments appeared to make F8C2PC an interesting memb rane solubilizing agent. Nevertheless, no protein in the F8C2PC extrac t could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octy l glucoside, some of the proteins, such as myeloperoxidase, could be a dsorbed land eluted), but not cytochrome b558 Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicle s and aggregates of small shapeless particles. Higher centrifugal forc es sedimented most proteins of the 100 000 g supernatant. As a check, the effect of F8C2PC was tested an sarcoplasmic reticulum vesicles, th e behavior of which with respect to the usual non-denaturating deterge nts has been well studied. There was little, if any, solubilization. W e conclude that, although supernatants of F8C2PC extracts of neutrophi l membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes ((C) Societe francaise de biochimie et biolog ie moleculaire / Elsevier, Paris).