Addition of glycerol during purification of banana (Musaceae, Musa cavendishii) pyrophosphate fructose 6-phosphate 1-phosphotransferase [(PFP), EC 2.7.1.90] initiated molecular aggregation of the enzyme. The aggregation process was dependent on the glycerol concentration. The native enzyme (66 kDa molecular mass) showed enhanced activity at 3% (V/V) or less of glycerol concentration. Glycerol concentration between 4 and 5% (V/V) affected a gradual and sequential aggregation of native form of the enzyme. These aggregated forms had molecular masses of 135, 200 and 270 kDa. The 135 and 200 kDa forms were stable for about 72 hrs and prolonged storage over 2 weeks resulted in the formation of the 270 kDa form. Concentration over 5% could reduce the time required for aggregation. Fru2.6 bis P activated the enzyme over ten fold, but did not help in the aggregation process. Studies on the role of glycerol on PFP specific activity suggested a difference in the activation process compared to that by Fru2.6bis P. Replacement of Hepes buffer by Tris increased the Fru2.6 bis P requirement for maximum activation by around 10 fold. Removal of glycerol from the buffer media resulted in almost complete inactivation of the enzyme.
Cellulose Chromatography, Affinity