Measurement of sidedness of isolated plasma-membrane vesicles: quantitation of actin exposure by DNase I inactivation.
A rapid and simple method is described to determine the orientation of isolated plasma membrane vesicle populations. The method is based on quantitation of the exposure of actin, an extrinsic membrane component bound exclusively to the cytoplasmic surface. Actin accessibility was determined using DNase I as a nonpermeating probe. The activity of this enzyme is lost upon complexation with actin, and inhibition can be conveniently monitored as the change in the rate of DNase-mediated hyperchromic reaction. Exposed actin is determined in intact vesicles, and total actin in detergent-lysed membranes; the ratio of these values equals the sum of inside-out and leaky vesicles. Sealed right-side-out vesicles are calculated by difference. Using thymocyte plasma membrane vesicles as a model system, we determined that 54% of the membrane is sealed in a right-side-out configuration. This value is consistent with independent determinations of inside-out membranes in the same preparation, obtained by 5'-nucleotidase latency measurements, and with reported values in other lymphocyte membrane preparations.
D000199:Actins / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D055598:Chemical Phenomena; D002621:Chemistry; D003593:Cytoplasm / Q000201:enzymology; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D066298:In Vitro Techniques; D008297:Male; D011817:Rabbits; D013499:Surface Properties; D013950:Thymus Gland / Q000648:ultrastructure
D000199:Actins; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I