On the Perturbation of Phospholipid Bilayers by Complement: The Use of Inside-Out Vesicles from Complement-Lysed Erythrocytes for the Demonstration of Perturbating Peptide Chains

It has been postulated by different groups that late complement components may partly insert into the phospholipid bilayer of complement lysed erythrocytes and form a trans-membrane channel.

The present report examines whether complement component antigens can be detected at the inner surface of complement lysed membranes by the use of inside-out vesicles. Insideout and right-side-out vesicles from osmotically or complement lysed sensitized sheep erythrocytes (EA or EAC1-9) were obtained by an incubation in a low ionic strength phosphate buffer, pH 8.0, followed by a gentle homogenization to liberate the entrapped vesicles.

Inside-out and right-side-out vesicles were separated on linear gradients of Dextran T 110 by centrifugation to equilibrium. Inside-out vesicles from EAC1-9 (IO-EAC) as well as those from osmotically lysed sensitized sheep erythrocytes (IO-EA) revealed both a very low binding of ³H-Ouabain in comparison to the respective right-side-out vesicles (RO-EAC and RO-EA). The four types of vesicles were radioiodinated by lactoperoxidase and glucose oxidase.