PURIFICATION, CHARACTERIZATION, AND CELLULAR-LOCALIZATION OF THE 100-KDA HUMAN PLACENTAL GTPASE-ACTIVATING PROTEIN

Human placenta contains, in addition to the ubiquitous p120-GTPase-act ivating protein (GAP), another isoform of 100 kDa, which is specific t o this organ. We have established a method for purifying this placenta l p100-GAP to near homogeneity. The purified p100-GAP allowed the prep aration of polyclonal and monoclonal anti Ras-GAP antibodies. Two mono clonal antibodies were selected for a two-site enzyme immunoassay. Thi s simple and accurate assay in turn facilitated the detection of the G APs 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. Ou r immunometric assay showed GAP to be present in human placental extra cts at the exceptional abundance of about 0.1% of the total protein co ntent. Quantitative assays showed p100-GAP to be up to 10 times more a bundant than p120-GAP. Use of our antibodies allowed the specific loca lization of placental GAPs to cytotrophoblasts and in the syncytiotrop hoblast barrier. Hence p100-GAP is shown to be found only in trophobla sts. The large quantity of p100-GAP in trophoblasts suggests that it m ay play a regulatory role in the proliferation or the differentiation of this cell type.