17] Isolation of glucose-1,6-diphosphate

Abstract

This chapter presents a procedure for the isolation of glucose-l,6-diphosphate by assay method in which the rate of the reaction catalyzed by phosphoglucomutase is proportional to the concentration of glucose-1,6-diphosphate. With this method it is possible to measure amounts of glucose-l,6-diphosphate ranging from 0.2 to 0.8 X 10 -3 micromole with an error of about 10%. Purification procedure in the method includes following steps: (1) preparation of the yeast extract, (2) precipitation with lead acetate, (3) destruction of fructose diphosphate, (4) barium fractionation, and (5) precipitation with acetone. It is suggested during precipitation that the treatment with H2S should be carried out rapidly, because glucose-l,6-diphosphate is acid-labile. The omission of step 2 does not give good results. During destruction of fructose the elimination of the inorganic phosphate with magnesia mixture in the usual manner results sometimes in a loss of about 40% of glucose-1,6-diphosphate by coprecipitation. Excess NH40H should be avoided because it interferes in the subsequent lead precipitation.