Purine Nucleoside Phosphorylase Activity of Blood. I. Erythrocytes 1

These investigations were undertaken to discover whether the membrane of the mature human erythrocyte is capable of metabolic activity that could possibly provide the means for active transport.Hoffman (2) has clearly shown that ATP provides energy for sodium transport, and it has been assumed that ATP for this process is supplied from within the erythrocyte. It seemed reasonable to examine membranes not only for possible sources of ATP production, but also for segments of metabolic pathways that could function as specific transport systems.Two methods have been in general use for the study of the enzymic composition of membranes (3). In one, whole cells are incubated with substrates or inhibitors to which the membrane is known to be impermeable. Any change in the external medium is then taken to represent enzymic activity present on the outer surface of the membrane. In the second method., labeled, permeable substrates are added to the medium, and after relatively short intervals, labeled compounds are extracted from the interior of cells and identified. The enzymic activity of the membrane is then deduced from the observed changes in substrates. Both methods have been useful, but neither can provide information about the organization of enzymes in the membranes. For this purpose, it is necessary to prepare and study erythrocyte membranes themselves.In the present study, morphologically intact homogeneous membranes, virtually free of hemoglobin, were prepared by dialysis of human erythrocytes against increasingly hypotonic saline * Supported by grants from the National Heart Institute (H-5305), U. S. Public Health Service, and the American Cancer Society (Institutional Grant 32A). Preliminary results of this study were submitted to the American Society for Clinical Investigation (1).tMarkle Scholar in Medical Science.solutions according to a modification of the nmethod of Danon, Nevo, and Marikovsky (4).Since mature human erythrocytes obtain their energy from glycolysis through generation of ATP, we examined erythrocyte membranes for pathways, or segments of pathways, of carbohydrate metabolism (Figure 1) that could be sources of energy for transport and perhaps also function as specific transport carriers. METHODS I. Preparation of membranes and hemolysatesA. Red-cell membranes. Fresh venous blood, drawn from healthy adult males or from patients hospitalized for nonsystemic illnesses (e.g., hernia repair, fractures), was immediately defibrinated with glass beads, transferred to 50-ml Lusteroid tubes, and centrifuged at 3,000 X g at 4' C for 5 minutes in a Lourdes LR refrigerated centrifuge with 9RA rotor. Serum and buffy coat were aspirated. Packed erythrocytes were washed by resuspension in 3 vol 0.145 M NaCl at 40 C and were centrifuged at 3,000 X g at 4' C for 5 minutes; they were washed a second time in 4 vol cold 0.145 M NaCl and centrifuged at 12,000 X g at 40 C for 15 minutes. The supernatant fluid was discarded, and samples of the packed cells were taken for counting in a Coulter model A counter. The a...

Section: Discussioncontrasting

confidence: 60%

Studies of the Metabolism of Human Erythrocyte Membranes*

Schrier¹

1963

“…phate per milliliter of incubation suspension When the nucleotides listed in Table VII (adeno-(Table VI). These latter observations are com-sine monophosphates, adenosine diphosphate, patible with the finding that erythrocyte nucleoside adenosine triphosphate, inosine-5'-phosphate) phosphorylase requires phosphate or arsenate for were employed, there was no enhanced protecits action (13,14). tion against osmotic lysis.…”

Section: Methodsmentioning

confidence: 87%

The Effects of Nucleosides On the Resistance of Normal Human Erythrocytes to Osmotic lysis12

Jaffé¹,

Lowy²,

Vanderhoff³

et al. 1957

The investigations reported in this paper proceeded on the hypothesis that maintenance of the structural integrity of the human erythrocyte is dependent on continued production and utilization of energy by the cell. In an attempt to test this hypothesis the susceptibility of fresh human erythrocytes to osmotic lysis was studied in terms of the influence of various compounds that might serve as substrates for energy yielding reactions within the erythrocyte. Particular attention was paid to glucose (3) and purine nucleosides (4, 7) which have been shown to prolong the viability of stored erythrocytes and to retard their progressive lysis and diminished resistance to hypotonic solutions. It may be noted, however, that the effectiveness of the purine nucleosides in the preservation of erythrocytes has recently been questioned (8 The concentrations of nucleosides were determined spectrophotometrically (9).In several experiments smears of the erythrocyte suspensions were prepared at different stages and stained with Wright's stain. No morphological differences be-1498

“…This suggests that a major route of AMP degradation does not pass through IMP, in contrast to previous reports (16,18). Rather, AMP may be hydrolyzed to adenosine (42), then deaminated to inosine (29,30,43,44), which finally undergoes phosphorolysis to hypoxanthine (27,28,45,46). Some of the hypoxanthine may then be incorporated into IMP (16, 18) (step 5), but the specific radioactivity of the IMP will be lower than that of the other compounds due to dilution with the unlabeled IMP present at the beginning of storage (Table I) or before labeled adenine appears as labeled hypoxanthine (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Action of Adenine in Red Cell Preservation*

Sugita¹,

Simon²

1965

Nakao and his associates have demonstrated that when blood, which had previously been stored at 40 C for 8 to 10 weeks in acid-citratedextrose (ACD), was subsequently incubated at 370 C with both adenine and inosine, regeneration of ATP and restoration of post-transfusion viability took place (1-3). ATP regeneration did not occur when the incubation medium contained either adenine or inosine alone. Moreover, supplementation of the ACD preservative with both adenine and inosine at the beginning of storage resulted in better maintenance of ATP and posttransfusion viability than in samples stored with inosine alone or not supplemented at all (3-5).Although these studies indicated that both inosine and adenine were necessary for rejuvenation of long-stored erythrocytes, it seemed possible that adenine could act without inosine in preventing loss of red cell viability when it was present from the beginning of storage, since at that time, unlike later in storage, glucose utilization for energy production is still intact. Moreover, eliminating inosine would have the further advantage of avoiding toxicity from uric acid overload in the recipient. Accordingly, we investigated the