Energy metabolism in human erythrocytes: the role of phosphoglycerate kinase in cation transport

A number of instantaneous changes occurred when picrate was added to a suspension of human red cells in stead)' state with respect to glycolysis and ion distribution across the membrane at pH 7.40. The rate of glycolysis increased, without change in glycolytic quotient, to a new steady-state value, the effect reaching a maximum of 1.75 times the rate of the control at 0.5 mM picrate. Inorganic phosphate (Pi) was released at a relatively constant rate, increasing with picrate concentration to 1.0 mmol Pl/liter cells x h at 5-6 mM picrate. The steadystate concentration of ATP and 1,3-diphosphoglycerate (1,3-DPG) decreased to new stable values within 15-45 rain after the addition of picrate. The ATP level was affected only at picrate concentrations of I mM or more, and the level of ATP stabilized at 75% of the control values at 4 mM of picrate. In contrast, 1,3-DPG concentrations decreased to 40% of the control value at 0.5 mM picrate. Higher concentrations of picrate resulted in only a small additional decrease in the stationary concentration of 1,3-DPG. A net efflux of cellular potassium at constant rate took place. This net efflux was an almost linear function of picrate concentration in the range 0.1-3 raM. At the latter concentration the net eff]ux amounted to about 2.7 meq/liter ceils x h and a further increase in picrate concentration caused only a minor increase in the potassium efflux. Possible mechanisms for the effects of picrate on human red cell glycolysis are discussed.

Section: Discussionmentioning

confidence: 70%

Accelerating effect of 2,4,6-trinitrophenol on the glycolytic rate of human red cells.

Vestergaard-Bogind¹,

Lunn²

1977

“…Finally, there may be proteins other than glycolytic enzymes that serve as a part of the ATP compartment. Similar schemes were proposed several years ago for functional relationships between the glycolytic enzymes and the Na/K pump (Parker and Hoffman, 1967;Proverbio and Hoffman, 1977;Schrier et al, 1975;Segel et al, 1975). More recently, such a multienzyme system was proposed on the basis of measurements of 31p nuclear magnetic resonances (NMR) of 2,3-DPG and G3P in red cells and bound to IOVs and on an effect ofouabain on the NMR (Fossel andSolomon, 1977 and1979;Solomon, 1978).…”

Section: Discussionmentioning

confidence: 89%

“…It has also been suggested that the ATP synthesized by membrane-associated GAPDH and PGK is compartmentalized in a "membrane pool," which is used preferentially by the Na/K pump (Hoffman and Proverbio, 1974;Okonkwo et al, 1975;Proverbio and Hoffman, 1977). Indirect evidence for the existence of a pump-associated ATP pool was provided by the inhibition of active transport in cells high in ATP, and depleted of triose phosphates (Sachs, 1972;Segel et al, 1975), and the inhibition of the use of a2P-labeled ATP by Na,Mg-ATPase after pretreatment with nonradioactive ATP (Proverbio and Hoffman, 1977). The suggestion of a membrane pool of ATP, however, has met with criticism.…”

Section: Introductionmentioning

confidence: 99%

Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes.

Mercer¹,

Dunham²

1981

204

ATP stimulates Na transport into inside-out vesicles (IOVs) made from human red cell membranes; strophanthidin inhibits the ATP-stimulated transport. The substrates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (glycolytic enzymes bound to the cytoplasmic surface of the red cell membrane) also stimulate Na transport into IOVs without added ATP. The elution of GAPDH from the membranes prevents the stimulation by the substrates, but not by exogenous ATP. la,~xo-kinase plus glucose (agents that promote breakdown of ATP) prevent stir, ulation of Na transport by exogenous ATP but not by the substrates for GAPDH and PGK.[32p]orthophosphate is incorporated into a membrane-bound organic phosphate compound shown chromatographically to be ATP. The level of membrane-bound ATP is decreased when Na is added, and this decrease is inhibited by strophanthidin. When further synthesis of [32p]ATP is blocked by the addition of unlabeled orthophosphate, all of the membrane-bound [32p]ATP is dissipated by the addition of Na. From these observations it was concluded that membrane-bound glycolytic enzymes synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump.

“…Finally, we can consider whether the ghost concentrations of ADP and ATP as referred to in this paper precisely reflect the concentrations of these nucleotides in the immediate proximity of the inner face of the Na/K pump . There is now substantial evidence (Parker and Hoffman, 1967;Okonkwo et al, 1975;Segel et al ., 1975 ;Proverbio and Hoffman, 1977 ;Mercer and Dunham, 1981) indicating that ATP (and ADP) may be compartmentalized within the regional domain of the pump. ATP pooled within this membrane compartment appears to be used preferentially with regard to cytoplasmic ATP as the proximate substrate of the pump .…”

Section: Discussionmentioning

confidence: 99%

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.

Kennedy¹,

Lunn²,

Hoffman³

1986

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from^-0 .2 to 50 were set and maintained (for up to 45 min) in this system . ATP and ADP concentrations were controlled by the addition of either a phosphoarginine-or phosphocreatine-based regenerating system . Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition . Na/K exchange was found to increase to saturation with ATP (K 1/ 2 99 250 juM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K112 a 350 juM). The elevation of ATP from -100 to 1,800 jM did not appreciably affect Na/Na exchange . In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange . The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na . Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E 2 " (K) " ATP --> E] -ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form . A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (EI). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E, P . (Na) . Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K. pump operates .