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Biochemical Studies of Human Placental Microvillous Plasma Membrane Proteins
Author(s) -
WEBB P.D.,
EVANS P.W.,
MOLLOY C.M.,
JOHNSON P.M.
Publication year - 1985
Publication title -
american journal of reproductive immunology and microbiology
Language(s) - English
Resource type - Journals
eISSN - 1600-0897
pISSN - 8755-8920
DOI - 10.1111/j.1600-0897.1985.tb00321.x
Subject(s) - transferrin , albumin , biochemistry , vesicle , syncytiotrophoblast , chemistry , polyacrylamide gel electrophoresis , coomassie brilliant blue , membrane protein , alkaline phosphatase , ovalbumin , microbiology and biotechnology , biology , membrane , staining , placenta , enzyme , pregnancy , fetus , genetics , immune system , immunology
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 (NH 4 SCN), the vesicles became smaller and more homogeneous. NH 4 SCN 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), IgH heavy chain (56kd), and Gc protein (56kd). Alpha‐2‐macroglobulin (α 2 M) was identified at 180kd and 95kd; the smaller component may be a proteolytic derivative indicating 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.