IN VITRO EXAMINATIONS OF PHOSPHATE EXCHANGE BETWEEN PLASMA AND ERYTHROCYTES IN THE BLOOD OF MULTIPLE SCLEROTIC PATIENTS

32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

“…tribution from the fast-labelling Pi pool is removed (Materials and methods section, Model B), the residual slow labelling of erythrocyte Pi (Figs. lb and 2b) strikingly resembles time courses reported in washed cells [9,10,26]. Furthermore, a recent study of renal epithelial cells, with no washing step, has demonstrated a rapid phase of labelling at low temperature, which was not attributed to trapped extracellular medium [29].…”

“…It has frequently been suggested that in erythrocytes there is compartmentation of nucleotides [6,8,10,26], glycolytic intermediates [27,28] and glycolytic enzymes [6], but nothing akin to the fast-labelling P1 pool has been reported. However, in many earlier studies, cells have been washed before P1 and 32p were measured [6,9,10,17,26]. In view of the rapid elution of the fastlabelling P, pool (Table lc), this may explain the discrepancy.…”

“…In human erythrocytes, flux from cellular Pi to organic phosphates occurs through the reaction catalysed by G3PDH in the Embden-Meyerhof pathway [7][8][9][10]. From the stoichiometry of this pathway, if this reaction were Assume that the fast-labelling pool (2t), which is a fraction F of total cellular Pi, exchanges so rapidly with extracellular P1 that it attains the same specific radioactivity as extracellular Pi before the cell suspension can be sampled.…”

“…Possibly, in the cell, G3PDH and a phosphatase are juxtaposed so that, in short incubations with [32P]Pi, G3PDH receives Pi of low specific radioactivity liberated from organic phosphate(s) by the phosphatase. In view of its large size [16,20], slow turnover [35] and slow labelling [6,9,26], the 2,3-BPG pool of the erythrocyte is clearly a good candidate for this organic phosphate.…”

“…There is currently considerable interest in the [32P]P, labelling of mammalian cells, for two reasons. Firstly, there is growing recognition of the importance of Pi in regulating cell function [1,2], even though there is uncertainty over whether Pi transport can be explained exclusively by direct transport through the membrane [3][4][5][6] or by an additional group translocation mechanism by glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC 1.2.1.12), incorporating extracellular Pi directly into cellular ATP [7][8][9][10]. Secondly, [32P]Pi labelling of intact cells is widely used in studying labelling of phosphoproteins and phospholipids (notably phosphoinositides).…”