ATP and Glycolytic Enzymes of Stored Blood ”Probable Cause of Loss of Phosphofructokinase during Storage”

1. To clarify the influence of pyruvate, adenine and inosine (PAI) to glycolytic enzymes of stored blood, the difference of erythrocyte enzyme activities between PAI-treated and plain ACD blood during storage was studied. As far as the glycolytic enzymes are concerned, phosphofructokinase (PFK), aldolase, trisephosphate isomerase, pyruvate kinase and lactate dehydrogenase all retarded loss of their activities in PAI-treated ACD blood than in plain ACD blood. Only hexokinase kept lower in PAI-treated ACD blood than in Plain ACD blood during 21 days of storage. PAI-treated ACD blood could preserve better ATP and lactate when incubation studies were performed on 21st day of storage than plain ACD blood could. These data probably indicate that PAI are effective to keep the glycolytic enzyme activities high, which may be advantageous for transfused erythrocytes to restore ATP level rapidly. 2. PFK activity decreased to about 20% and 35% of original level on 28th day of storage in plain and PAI-treated ACD blood, respectively. However, the loss of PFK activity recoverd up to original level when stored blood was incubated with adenine and inosine in response to ATP regeneration. In addition, PFK activity of stored blood remained original level when they were hemolized with a special solution (A. solution) which contained ATP, ammonium sulfate, mercaptoethanol, EDTA and tris-phosphate (pH 8.0). Among these reagents, mercaptoethanol was the most effective to restore the loss of PFK activity during storage. On the other hand, PFK activity was completely inhibited with p-chloro-mercuribenzoic acid and its inhibition did not occur when mercaptoethanol was present. In addition, PFK activation by mercaptoethanol was greater in stored blood than in fresh blood. These data suggest that the loss of PFK activity of stored blood is produced by affecting SH group at the catalytic site of PFK, probably by oxidation, and the PFK activiy is reversible when stored blood return to more physiological condition accompanied with restored red cell metabolism probably through regaining SH groups of PFK.