PURIFICATION OF AN APOLIPOPROTEIN-A BINDING-PROTEIN FROM MOUSE ADIPOSE-CELLS

A protein recognizing apolipoproteins A(I), A(II) and A(IV) was purified from cultured mouse adipose cells of the Ob17MT18 clonal line. Apolipoprotein A binding sites were solubilized in the presence of proteinase inhibitors using the non-denaturating detergent CHAPS. Chromatography of the solube extract on DEAE-trisacryl was followed by immunoaffinity chromatography of the complex apolipoprotein A1-binding proteins on anti-(apoliprotein A(I)) coupled to Sepharose 4B and then by h.p.l.c. on an RP-Select B column. A 1400-fold purification over the starting crude homogenate was achieved. The purified material contained two proteins that were both able to bind apolipoproteins A(I), A(II) and A(IV), but not low-density lipoprotein. Glycopeptidase F treatment showed the existence of a single protein bearing either N-linked high-mannose or complex oligosaccharide chains. The purified material showed an apparent molecular mass of 80 ± 9 kDa by h.p.l.c. on a TSKG 3000 SW column. Rabbit polyclonal antibodies directed against the purified material revealed two protein bands of 80 and 92 kDa after SDS/PAGE under reducing conditions and immunoblotting. These bands were undetectable in growing Ob17PY cells previously shown not to bind the various apoliproteins A and not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Ob17PY cells which have recovered these properties.