Abstract
Phosphorylation of Pyruvate by the Pyruvate Kinase Reaction and Reversal of Glycolysis in a Reconstructed System
Highlights
MethodsATP and ADP were assayed by the method of Bowen and Kerwin (15) after removal of Mg ++ from the trichloroacetic acid filtrates of reaction mixtures by passing them through a column of Dowex 50-X2, H+ form
Pyruvate is phosphorylated by reversal of the pyruvate kinase reaction
It will be noted that PE accumulation is strongly dependent on the presence of lactic dehydrogenase; reoxidation of DPNH formed in the glyceraldehyde-3-P dehydrogenase reaction is necessary to maintain a high level of 1,3-diphosphoglycerate which in turn, via the phosphoglycerate kinase reaction, permits a high ATP:ADP ratio required for reversal of the pyruvate kinase reaction
Summary
ATP and ADP were assayed by the method of Bowen and Kerwin (15) after removal of Mg ++ from the trichloroacetic acid filtrates of reaction mixtures by passing them through a column of Dowex 50-X2, H+ form. Removal of Mg++ is necessary since it interferes with the subsequent assay. Mitochondrial Preparations-These were made from rat liver by the method of Hogeboom (16) except that the suspending medium used was 0.25 1~ mannitol containing 1 mg. Mannitol was used instead of sucrose because contamination of some yeast enzyme preparations with invertase (which catalyzed glucose formation) aud hexokinase led to net esterification of Pi, i.e. glucose 6-phosphate formation, without the addition of pyruyate as a phosphate acceptor.
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