Sequential preparation of highly purified microvillous and basal syncytiotrophoblast membranes in substantial yield from a single term human placenta: inhibition of microvillous alkaline phosphatase activity by EDTA

Abstract

The human placental syncytiotrophoblast is a highly polarised epithelial layer responsible for regulating materno-fetal exchange. We here describe a novel procedure for isolating paired fractions of the maternal-facing and fetal-facing plasma membranes from this syncytium, from a single placenta, without the need for homogenisation procedures. This reduces the potential for contamination of these membrane fractions by intracellular membranes, or from plasma membranes from other cell types within the placenta. Microvillous membrane vesicles (MVM) were obtained by gentle stirring of dispersed villous tissue. The tissue sedimented at the end of this procedure was subjected to sequential ultrasonication to release the basal membrane (BM). Crude MVM was subsequently purified on a discontinuous sucrose gradient. Crude BM was further purifed using either discontinuous Ficoll or sucrose gradients. The Ficoll procedure, while producing a BM fraction extremely enriched in marker enzyme, resulted in unacceptably low protein recoveries and hence the sucrose gradient procedure was also adopted for BM. Yields for MVM and BM produced on sucrose density gradients approached 30 mg/100 g tissue. The MVM fraction was composed of vesicles of 232 ± 9 (S.E.) nm diameter of which nearly 90% were ‘right side out’. These membranes were 37-fold enriched in the marker enzyme alkaline phosphatase. Purified BM vesicles were 317 ± 14 nm in diameter, also approximately 90% ‘right side out’ and over 40-fold enriched in dihydroalprenolol binding. Cross-contamination or contamination from intracellular membranes was negligible. MVM alkaline phosphatase activity was shown to be inhibitable in a dose-and time-dependent manner by EDTA present in the storage buffer.