Vesicles with transport capability isolated from cultured fibroblasts.

Abstract

Plasmalemmas from cultured human skin fibroblasts, isolated by a simple and reproducible method, can be converted to vesicles which are capable of active transport of aminoacid when glutathione is included within the vesicles. In the isolation, the plasmalemmas are stabilized with a Ricinus lectin, with preservation of the classic plasmalemma enzymes. The procedure has been applied successfully to a number of normal and abnormal human skin fibroblasts including those of myotonia dystrophica and progeria victims, and to the lung fibroblast WI-38. As part of a study of the characteristics of transport enzymes related to aging and to the muscular dystrophies in cultured fibroblasts, it was desirable to simplify the system by the use of vesicles prepared from the fibroblast plasmalemmas. The procedure described below is similar in some respects to that applied to another membranous system in the use of a lectin to stabilize the plasmalemma structure. The use of other stabilizing agents such as heavy metals and surfactant polymers which react with the membranes, but could compromise the reliability of the enzyme assays, was avoided. Since the focus of this study was on the enzymic systems of transport, the examination of facilitated diffusion or exchange was excluded. The well defined glutathione-dependent mechanism of aminoacid transport was examined to verify the competence of the vesicles for active transport and to confirm their sidedness. Other enzymes of transport, the ATPases, and membrane marker enzymes were also determined.