Year-end Sale % OFF 
Abstract
Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.
Highlights
An alkyl substituent (&sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer
37 “C, approximately three-quarters of the &sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h
The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and Mycoplasmas are the only organisms containing a single membrane and no cell wall that are capable of autonomous growth [1]
Summary
Cholesterol, desmosterol, bovine serum albumin (Fraction V, fatty acid poor), oleic and palmitic acids, and deoxyribonuclease were obtained from Sigma. &Sitosterol (referred to as sitosterol below) and stigmasterol were from Steraloids (Wilton, NH). Desmosterol was purified by preparative thin layer chromatography using Silica Gel Hplates (Analtech, Newark, DE) developed with diethyl ether/petroleum ether (31, v/ v). The series of cholesterol analogs in which the length of the side chain varies and theterminal methyl branch is absent (2O(R)-n-alkylpregn5-en-3P-01s)was generously supplied by Dr Toshiyuki Akiyama, University of Tokyo. These analogs, which are referred to by the total number of carbon atoms they contain [14, 15], were synthesized, characterized, and shown to be pure by Morisaki et al [16]. Labeled sterols were analyzed for radiochemical purity by counting zones scraped from thin layer plates (silica gel, Analtech); the radiochemical purity of all of the sterols was 398%
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
