Purification of an apolipoprotein A binding protein from mouse adipose cells.
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.
Barbaras
R
R
Centre de Biochimie du CNRS, Faculté des Sciences, Nice, France.
Puchois
P
P
Fruchart
J C
JC
Pradines-Figueres
A
A
Ailhaud
G
G
D000180:Acrylic Resins; D000273:Adipose Tissue / Q000032:analysis* / Q000166:cytology; D000581:Amidohydrolases; D000818:Animals; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins / Q000302:isolation & purification*; D001054:Apolipoproteins A / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification*; D008078:Cholesterol, LDL / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding
D000180:Acrylic Resins; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins; D001054:Apolipoproteins A; D002352:Carrier Proteins; D008078:Cholesterol, LDL; C022205:apolipoprotein A-IV; C049689:DEAE-Trisacryl; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase