guanosine-triphosphate and alpha-glycerophosphoric-acid

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2, 6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.

Topics: Animals; Binding Sites; Chickens; Circular Dichroism; Diethyl Pyrocarbonate; Enzyme Inhibitors; Escherichia coli; Ethylmaleimide; Fructosediphosphates; Glycerophosphates; Guanosine Triphosphate; Kinetics; Liver; Multienzyme Complexes; Mutagenesis, Site-Directed; Peptide Fragments; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Structure, Secondary; Rats; Recombinant Proteins; Sequence Deletion; Spectrometry, Fluorescence

It has been proposed that the alpha-glycerophosphate (alpha-GOP) shuttle plays a crucial role in regulation of glycolysis in beta-cells by linking reoxidation of cytosolic NADH to formation of ATP in the electron transport chain (J. Biol. Chem. 265: 8287, 1981). Direct evidence for this suggestion is still lacking, however. In this work the operation of the alpha-GOP shuttle was investigated in the insulin-secreting cell line HIT-T15. The constituent enzymes of the pathway were found to be present in HIT cells. Flavin-linked alpha-GOP dehydrogenase was associated with the mitochondrial fraction, whereas NAD+-dependent alpha-GOP dehydrogenase was localized in the cytosol. In the presence of amobarbital (used to preserve the function of the alpha-GOP shuttle under conditions where oxidation of NADH by the respiratory chain was blocked), glucose increased insulin secretion, O2 consumption, and the cell [ATP]/[ADP] when compared with amobarbital alone. These results indicate that the alpha-GOP shuttle contributes to ATP generation in HIT cells and that its activation may be necessary for the initiation of insulin secretion by glucose.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amobarbital; Cell Line; Clone Cells; Cytosol; Electron Transport; Glucose; Glycerolphosphate Dehydrogenase; Glycerophosphates; Glycolysis; Guanosine Diphosphate; Guanosine Triphosphate; Insulin; Insulin Secretion; Islets of Langerhans; Lactates; Lactic Acid; NAD; Oxidation-Reduction; Oxygen Consumption; Pyruvates; Pyruvic Acid