Figure 1. (A) Native PAGE analysis of adiponectin oligomeric state following reduction and collapse of octadecamers and subsequent removal of reductant by dialysis.

Purified bovine serum octadecamers were collapsed to trimers in 100 mM DTT or 200 mM βME and subsequently dialyzed against PBS over a 3 hr period to remove DTT or βME. Following fractionation in PAGE, the proteins were detected by fluorescence from an infrared stain as described in Experimental Procedures. Two samples, one in DTT and the other in βME, were not dialyzed against PBS to assess if re-oligomerization to octadecamers depended upon removal of reducing agents. (B) Densitometric analysis of adiponectin octadecamer (top panel), hexamer (middle panel), and trimer (bottom panel) levels during removal of reductant by dialysis following collapse of purified octadecamers. The integrated intensity of each band corresponding to a specific oligomer at a particular time point was normalized to the sum of the integrated intensities of all three major oligomers at the same time point to obtain a value for the percentage of total adiponectin. Average percentage values and standard error of the mean from four and five independent experiments whose initial reductant was, respectively, DTT (open circle) and βME (closed square) were plotted. (C) Oligomerization state of adiponectin following titration with different concentrations of DTT. Purified octadecamers were treated with 1, 10, and 100 mM DTT and resulting products’ oligomerization states were analyzed using native PAGE as described above and in Experimental Procedures. (D) Effect of pH and βME concentration on adiponectin oligomerization state. Adiponectin octadecamers were treated with 2, 20, 50 or 200 mM βME at pH 5 or 7 as described in Experimental Procedures. Native PAGE was used to analyze oligomerization states following the treatments.