title: Purification, characterization, and cellular localization of the 100-kDa human placental GTPase-activating protein.
abstract: Human placenta contains, in addition to the ubiquitous p120-GTPase-activating protein (GAP), another isoform of 100 kDa, which is specific to this organ. We have established a method for purifying this placental p100-GAP to near homogeneity. The purified p100-GAP allowed the preparation of polyclonal and monoclonal anti Ras-GAP antibodies. Two monoclonal antibodies were selected for a two-site enzyme immunoassay. This simple and accurate assay in turn facilitated the detection of the GAPs during purification. The purified p100-GAP has a specific activity identical to and catalytic properties similar to those of native p120-GAP. Sequence analysis of p100-GAP revealed almost total identity to the known corresponding sequences predicted by the cDNA. The purified p100-GAP kept its activity for 1 year when stored at -80 degrees C. Our immunometric assay showed GAP to be present in human placental extracts at the exceptional abundance of about 0.1% of the total protein content. Quantitative assays showed p100-GAP to be up to 10 times more abundant than p120-GAP. Use of our antibodies allowed the specific localization of placental GAPs to cytotrophoblasts and in the syncytiotrophoblast barrier. Hence p100-GAP is shown to be found only in trophoblasts. The large quantity of p100-GAP in trophoblasts suggests that it may play a regulatory role in the proliferation or the differentiation of this cell type.
authors.affiliation: Unité 245 Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: G
authors.initials: G
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Mollat
authors.forename: C
authors.initials: C
authors.lastname: Carles
authors.forename: M
authors.initials: M
authors.lastname: Riva
authors.forename: Y
authors.initials: Y
authors.lastname: Frobert
authors.forename: A
authors.initials: A
authors.lastname: Malassiné
authors.forename: W
authors.initials: W
authors.lastname: Rostène
authors.forename: D C
authors.initials: DC
authors.lastname: Thang
authors.forename: B
authors.initials: B
authors.lastname: Beltchev
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D020690:GTPase-Activating Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010920:Placenta / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D014018:Tissue Distribution; D014327:Trophoblasts / Q000737:chemistry; D020703:ras GTPase-Activating Proteins
chemical_list: D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; D011506:Proteins; D020703:ras GTPase-Activating Proteins