Purification of an apolipoprotein A binding protein from mouse adipose cells

• Published in: Biochemical journal Vol. 269; no. 3; pp. 767 - 773
• Main Authors: Barbaras, R, Puchois, P, Fruchart, J C, Pradines-Figueres, A, Ailhaud, G
• Format: Journal Article
• Language: English
• Published: England 01.08.1990
• Subjects: Acrylic Resins; adipocytes; Adipose Tissue - analysis; Adipose Tissue - cytology; Amidohydrolases; Animals; Apolipoprotein A-I; Apolipoprotein A-II; Apolipoproteins - isolation & purification; Apolipoproteins A - metabolism; Binding Sites; Carrier Proteins - isolation & purification; Cholesterol, LDL - metabolism; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange - methods; Humans; Mice; Molecular Weight; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Protein Binding
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2690767

A protein recognizing apolipoproteins AI, AII and AIV 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 soluble extract on DEAE-Trisacryl was followed by immunoaffinity chromatography of the complex apolipoprotein AI-binding proteins on anti-(apolipoprotein AI) 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 AI, AII and AIV, 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 apolipoproteins A and not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Ob17PY cells which have recovered these properties.