Biochemical studies of human placental microvillous plasma membrane proteins.
Isolated human syncytiotrophoblast microvillous plasma membranes (StMPM) have been examined by electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional PAGE (2D-PAGE), and immunoblots. Electron microscopy of StMPM pellets revealed populations of membrane-bounded vesicles that disrupted after treatment with the chaotrope 3M KCl for 16 hr; with increasing molarity of another chaotrope (NH4SCN), the vesicles became smaller and more homogeneous. NH4SCN treatment resulted in significant reduction on SDS and 2D-PAGE analysis of only one protein at 80kd, shown by immunoblotting to be transferrin; 3M KCl had little effect and appeared to be a poor chaotrope. Chromogenic silver staining of SDS-PAGE gels demonstrated over 50 StMPM-associated discrete protein components. Immunoblotting revealed transferrin (80kd), albumin (65kd), IgG heavy chain (56kd), and Gc protein (56kd). Alpha-2-macroglobulin (alpha 2M) was identified at 180kd and 95kd; the smaller component may be a proteolytic derivative indicating alpha 2M binding to a trophoblast surface protease. Numerous discrete protein dots, and groups of dots characteristic of charge heterogeneity of individual proteins, were observed on high resolution 2D-PAGE. The most intensely stained proteins were transferrin (80kd), albumin (65kd), placental-type alkaline phosphatase (66kd), and actin (46kd). This 2D-PAGE technique is a superior method for analyzing the trophoblast membrane proteins, and the system described will enable systematic mapping of these components.
D000418:Albumins / Q000302:isolation & purification; D002462:Cell Membrane / Q000032:analysis; D002824:Chorionic Villi / Q000032:analysis* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007120:Immunochemistry; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008970:Molecular Weight; D011247:Pregnancy; D014168:Transferrin / Q000302:isolation & purification
D000418:Albumins; D008565:Membrane Proteins; D014168:Transferrin