Studies of the Metabolism of Human Erythrocyte Membranes*

Abstract

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...

Recoupling the Na-K Pump

“…Feig et al (6) proposed that ATP derived from the PGK reaction may be preferentially used for the active Na outflux since the ouabainsensitive Na outflux fell at a time when triose phosphate, the source of substrate for the PGK reaction, was low but the total intracellular ATP + 2,3-DPG was still high. Since part of the red cell PGK is membrane bound (16), this would fit with the findings of Parker and Hoffman (14) and Proverbio and Hoffman (15). Since it is clear that only a fraction of the ATP consumed by the red cells is used for cation transport, there is no way of telling from the present experiments whether or not ATP from the PGK reaction is preferentially used by the pump.…”

Enzymatic degradation of uric acid by uricase-loaded human erythrocytes.

“…If enzyme-loaded cells are to survive in vivo, however, they must be able to use glucose as the energy source and have an intact glycolytic pathway. Schrier (11) has shown that in extensively hemolysed erythrocyte ghosts, the level of all the glycolytic enzymes is extremely low. However, we would expect that if the cells are kept concentrated during hemolysis by the addition, for example, of only 4 vol water to 1 vol cells, a substantial residual level (.…”

Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles

“…Kant & Steck (1973) report that binding of the enzyme is also influenced by micromolar amounts of NAD+, NADH and glyceraldehyde 3-phosphate. In a previous paper (Schrier, 1963;Kant &Steck, 1973;Vol. 159 MacDaniel et al., 1974 concentration -of free enzyme but decreases as the ionic strength of the incubation medium is raised by the addition of KCI.…”