The role of inositol phosphates in the signal transduction of AKH - I , - II and - III in the locust fat body

..................................... 5 Introduction................................ 9 1.1 The fat body ................................................................... 9 1.2 The adipokinetic hormones .......................................... 9 1.3 Glycogen phosphorylase ............................................. 10 1.4 The role of cyclic AMP ............................................... 10 1.5 The role of intraand extracellular calcium ..............11 1.6 The role of inositol phosphates .................................. 13 Materials and Methods............ 15 2.1 Animals ....................................................................... 15 2.2 Chemicals .................................................................... 15 2.3 Preparation of fat body ............................................... 16 2.4 Total InsP measurement in the fat body .................... 16 2.5 Determination of salt concentrations needed for inositol phosphates elution ........................................... 17 2.6 Measurement of individual InsP in the fat body ....... 17 2.7 Measurement of radioactivity ..................................... 18 2.8 Data interpretation InsP measurements .................... 18 2.9 Measurement of Ins(1,4,5)P3 levels in the fat body ... 18 2.10 Protein determination ............................................... 19 2.11 Involvement of phospholipase C in glycogen phosphorylase activation .............................................. 20 2.12 Glycogen phosphorylase assay ................................. 20 2.13 Involvement of phospholipase C in InsP formation 20 Results ....................................... 22 3.1 The effect of AKH on the formation of total InsPs ... 22 3.2 Determination of elutionbuffer concentrations of individual InsPs ............................................................ 24 3.3 The effect of AKH on the formation of the total InsPs .............................................................................. 26 3.4 Ins(1,4,5)P3 determination using a radio-immuno assay .............................................................................. 28 3.5 Role of PLC in AKH-induced InsP formation and glycogen phosphorylase activition................................ 29 Discussion and Conclusions .... 32