Stored, depleted RBC were rejuvenated with respect to their levels of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and P50 by acid-citrate-dextrose perservatives containing phosphoenolpyruvate (PEP) without sucrose. The restorations of P50 and 2,3-DPG were dependent on the phosphoenolpyruvate concentration. Erythrocyte P50 and 2,3-DPG, even after treatment with these preservatives, decreased with increasing storage period, but the P50 and 2,3-DPG of five-week-old blood were still higher than the corresponding values of fresh blood. ATP concentration was also increased by treating stored blood with preservatives containing phosphoenolpyruvate, but the elevated ATP of five-week-old blood was only about 50 percent of fresh blood. The ATP level could not be raised further by increasing phosphoenolpyruvate concentration but was improved by supplementation with adenine and nucleosides. Incubation of stored blood with 15 mM phosphoenolpyruvate was sufficient to restore ATP, 2,3-DPG and P50 of three-week-old blood to nearly normal. The results of these studies indicate that sucrose is not necessary for PEP to be effective as a preservative additive.

Phosphoenolpyruvate was found to be transported across the erythrocyte membrane by a carrier-mediated transport system. The transport of phosphornolpyruvate was competitively inhibited by inorganic phosphate (4 = 24 mM) and pyridoxal 5-phosphate (4 = 0.2 mM), whcrcas the transport was noncompetitively inhibited by L(+)-lactate (y = 37 mM). Specific inhibitors for the inorganic anion transport system such as 4,4-diisothiocyanostilbene-2,2'-disulfonic acid and 4,4-dinitrostilbene-2,2'-disulfonic acid strongly inhibiied the phosphoeiiolpyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal5-phosphate and NaBH,. Transport activities of phosphoenolpyruvate and inorganic phosphate in the treated cells were similarly inhibited by pyridoxal 5-phosphate depending on its concentrations. 4,4-Dinitrostilbene-2,2'-disulfonic acid protected both transport activities against the pyridoxal 5-phosphate/NaBH4 treatment.The major integral membrane protein, band 3, was preferentially labelled by treating erythrocytes with pyridoxal 5-phosphate and NaB[3H]H,. The radioactive incorporation into band 3 was confirmed by two-dimensional gel electrophoresis combining isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel clectrophoresis in the second dimension.Taken together, these results suggest that band 3 mediates the transport of phosphoenolpyruvate as well as inorganic phosphate.Organic phosphate compounds have generally been regarded as impermeable to the erythrocyte mernbranc. We have previously shown, however, that in the presence of impermeable solutes, such as sucrose or citrate, phosphocnolpyruvate-(P-pyruvate) is able to penetrate the erythrocyte membrane by a carrier-mediated transport system [I]. The transport appears to be specific to P-pyruvate among the phosphorylated glycolytic intermediates. 2-Phosphoglycerate, whose molecular weight and pK values are quite similar to those of P-pyruvate, is impermeable to the cell membrane. The P-pyruvate transport is dependent on pH, osmolarity and the concentration of sucrose or citrate in the incubation media, and is inhibited by 4-acetamido-4-isothiocyanostilbenc-2,2'-disulfonic acid (SITS) or pyridoxal 5-phosphate (pyridoxal-P)/NaBH, treatment [l, 21. Although P-pyruvate is transported against a concentration gradient, the transport is characterized as passive and the apparent uphill transport is explained by the Donnan equilibrium [I].The P-pyruvate transported into erythrocytes is metabolized rapidly to 2,3-bisphosphoglycerate and pyruvate [3 -51. Such evidence has been utilized for development of a new procedure to increase 2,3-bisphosphoglycerate and ATP concentrations in depleted, stored erythrocytes [6-81. The trans-The preliminary report of this work was presented at the Tokai University Symposium on Biomembranes, Vedbaek, Denmark, June 14- 15, 1982.Ahhreviutions. P-pyruvate, phosphoenolpyruvatc; pyridoxal-P, pyridoxal 5-phosphate; SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfoiiic ac...