Biomedical subjects
Publications and source records attributed to W E Marshall.
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Erythrocytes from patients with Duchenne's and myotonic muscular dystrophies contained more adenosine triphosphate (ATP) and produced more lactate than did normal erythrocytes in incubation studies conducted in vitro at an initial pH of 7.4. Since the same results were obtained in two different genetic dystrophies, these metabolic variations appear to be secondary to the primary changes occurring in these diseases. Following ouabain treatment, ATP content increased and lactate production decreased in erythrocytes from both dystrophies. This result differs from one reported earlier in experiments conducted at alkaline pH.
The addition of 3 mM hemoglobin to 1-10 mM ATP solutions at pH 6.75 resulted in a linear relationship between the change in chemical shift (deltadelta) of the gamma-phosphate of ATP and the percent ATP bound to hemoglobin. The data points obtained with oxyhemoglobin and deoxyhemoglobin fell on the same straight line. In the presence of 3 mM Mg2+, the delta delta decreased curvilinearly as the percent ATP bound was raised. In this case, the percent ATP bound to deoxyhemoglobin was greater than to oxyhemoglobin at the same delta delta value, indicating that the extra ATP binding occurs through groups other than phosphate.
Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.
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1. The chemical shifts (delta) of the phosphates of 2,3-diphosphoglycerate and adenosine triphosphate (ATP) were determined by phosphorus nuclear magnetic resonance (31P NMR) spectroscopy and were found to be displaced downfield following the addition of hemoglobin (3 mM) to a solution of either diphosphoglycerate (5 mM) or ATP (1 mM). 2. The binding of these compounds to hemoglobin was also determined by membrane ultrafiltration. A direct relationship was observed between the change in chemical shift ((delta delta) of the 2-P and 3-P of diphosphoglycerate and the percent diphosphoglycerate bound, when the latter was varied by altering pH, oxygenation state, or total diphosphoglycerate concentration. 3. In comparable studies with ATP binding, a linear relationship between the delta delta values of the gamma-, beta-, and alpha-P of ATP and the percent of ATP bound was not observed when the data from all of the experiments were plotted. NMR signals were not detectible in deoxyhemoglobin solutions containing 1 mM ATP but were seen in solutions containing 3.8 mM ATP. 4. The results indicate that 31P NMR spectroscopy is a promising tool for investigating organic phosphate interactions with hemoglobin.
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Free ATP and DPG concentrations in ACD-preserved human erythrocytes (rbc) were estimated by measuring these organic phosphates in ultrafiltrates of cell lysates prepared at 0 to 2 C. The free ATP concentration in hypotonic hemolysates decreased from 0.59 to 0.08 mumol/ml rbc during four weeks of cold storage. In French Pressure Cell lysates, free ATP increased from 0.23 to 0.47 mumol/ml rbc in the first week and then declined to 0.12 mumol/rbc during the following three weeks. In two weeks, the free DPG level decreased from 0.64 to 0.41 mumol/ml rbc in hypotonic lysates and from 1.08 to 0.15 mumol/ml rbc in French Pressure Cell lysates. It is proposed that, as total DPG concentration decreases during cold storage, free ATP concentration also decreases due to increased ATP binding to hemoglobin sites vacated by DPG. It is suggested that it is the change in the free rather than the total ATP which may be more relevant to the metabolism of the cold-stored erythrocyte.