Purification, characterization, and cellular localization of the 100-kDa human placental GTPase-activating protein.
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.
Zhang
Y
Y
Unité 245 Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.
Zhang
G
G
Mollat
P
P
Carles
C
C
Riva
M
M
Frobert
Y
Y
Malassiné
A
A
Rostène
W
W
Thang
D C
DC
Beltchev
B
B
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
D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; D011506:Proteins; D020703:ras GTPase-Activating Proteins