Purification, characterization, and cellular localization of the 100-kDa human placental GTPase-activating protein.

Y Zhang,G Zhang,P Mollat,C Carles,M Riva,Y Frobert,A Malassiné,W Rostène,D.C Thang,B Beltchev

doi:10.1016/s0021-9258(17)46708-1

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.

Highlights

Human placenta contains, in addition to theubiquitous pl20-GTPase-activating protein (GAP), another iaoformof 100kDa, which is specific to thisorgan
The polynucleotide binding will bediscussed below.p100GAPwas purified to near homogeneity on a CMSepharose column (Fig. 1, lane 6, and Fig.[2], lane 6 ), the protein content in the 0.23 M eluted fraction being quite close to the p100-GAP content as determined by an immunometric assay (Table I)
Measurement of GAP during the purification was highlyfacilitated by the use of a two-site enzyme immunometric assay instead of the measurement of GAP activity

Summary

Recipient of a predoctoral scholarship from the MinisGre de la

Vert) from the Institut National de la Sa& et de la Recherche MBdicale. domain of 120-GAP (18-20) and exhibits the same catalytic activity. Peptide Sequence Analysis-Purified p100-GAP was digested for 18 h at 35 "C with the Lys-specific endoproteinase in a buffer containing 50 mM Tris/HCl, pH 8.0, 10% glycerol (v/v), 1mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride with a protease to negativeregulators of Ras-GTP or as downstream effector protein ratio of 150 (w/w). 0.2 mM phenylmethylsulfonyl fluoride, 10% (v/v) glycerol) at 4 "C for The mouse monoclonal antibody was used at a dilution of 20 pg/ 2 h with one change of buffer, and applied to a poly(1)-Sepharose ml. Column (1.6 X 12 cm, 0.3mgof poly(I)/ml) This column was first Methods specificity was controled by substitution of the primary washed with buffer B containing 0.15 M KCI, and p100-GAP was eluted with 0.28 M KC1 (flow rate = 3 ml/min).

RESULTS

Total GAP

GYLLK EISMEDEATTLFAA

DISCUSSION