31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system.

Procedural modifications of the luciferase method for ATP assay in conjunction with enzymatic conversion of AMP and ADP allow the assay of all three adenine nucleotides in quantities ranging from 4 to 20 pmoles. An unmodified Beckman scintillation detector at ambient temperature and in a coincidence mode of operation serves as a suitable instrument for quantitating light emitted by the enzyme preparation. The most significant modifications include use of Ca₃(PO₄) activated crude arsenate extracts of desiccated firefly lanterns, low arsenate concentrations during the assay, and an assay pH of 8.0. Extracts handled in this manner exhibit approximately fivefold higher activity than nonactivated extracts employed at pH 7.4 and 50 mm arsenate. Stability of activated extracts is also somewhat greater than for nonactivated preparations. ADP can be 95% enzymatically converted to ATP by treatment with phosphoenolpyruvate and pyruvate kinase under the conditions described. If myokinase is included, approximately 90% of sample AMP can be converted to ATP. Follwing the appropriate enzymatic treatment, the nucleotides are assayed as ATP and amounts calculated by comparison to curves established for known nucleotide standards. The method is appropriate for perchloric acid extracts of biological tissue and certain considerations necessary for application to experimental situations are described.     

A method for determination of glucose 1,6-bisphosphatase

A sensitive and specific method to measure glucose 1,6-bisphosphatase activity, which allows the identification of the reaction products is described. [U-14 C]Glucose 1,6-P2, synthesized by the glucose 1-P kinase activity of phosphofructokinase, is used as substrate. The reaction products are separated and identified by chromatography on ion-exchange paper.     

Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase,glucose 6-phosphate dehydrogenase,malate dehydrogenase,and lactate dehydrogenase

The activities of four enzymes were determined during the first four days of mouse embryogenesis. Two enzymes, fructose 1,6-diphosphate aldolase and malate dehydrogenase, increase about 30% in activity, and this increase is attributed to slow but continued enzyme synthesis. The other two enzymes, glucose 6-phosphate dehydrogenase (X-linked) and lactate dehydrogenase, remain constant for the first two days and then decline exponentially with half-times of 19 and 17 hr, respectively. These declines in activity cannot be explained by the appearance of soluble inactivators or by the disappearance of soluble activators. Likewise, although temporally related to the passage of the embryos from the oviducts into the uterine horns, the changes in enzyme activity do not result from this change in embryonic environment, and specific degradative processes beginning on day 2 of embryonic development are postulated.This study was supported by USPHS Grant HD 03132 and by a grant from the School of Medicine, University of California, San Francisco Medical Center. The senior author is the recipient of USPHS Research Career Development Award HD 35,565.     

A model for metalloprotein-catalysed ADP, ATP transport in mitochondria

We propose a hypothetical model for the transmembrane exchange reaction catalysed by the mitochondrial adenine nucleotide carrier protein, which basically consists of an alternating reorientation of a transitory carrier—metal—nucleotide complex. The key features of the model are: the participation of an intrinsic divalent metal ion in the course of transport catalysis; the different stability constants of protonated and deprotonated nucleotide—metal complexes; the exposure and retraction of strategic arginyl residues; the alternating reorientation of the active center involving a change from the cytosolic conformation (C_c) to the matrix conformation (C_m).     

Change in the content of ATP and 2,3-diphosphoglycerate in the erythrocytes of rats adapted to hypoxia]

It was shown that on the 30th-60th days of training rats to hypoxia under conditions of pressure chamber there was an increase in ATP and 2,3-diphosphoglycerate content in erythrocytes. By changing the affinity of hemoglobin to oxygen the mentioned shifts could play an important role in the improvement of oxygen supply to the tissues.     

The effects of ionophore A23187 on erythrocytes relationship of ATP and 2,3-diphosphoglycerate to calcium-binding capacity

The divalent cation ionophore, A23187, was employed as a means to load fresh human erythrocytes with calcium, and the capacity for accumulation was characterized. Erythrocytes exposed to A23187 in calcium-containing media rapidly accumulated calcium in millimolar quantities. The final cellular concentration was dependent upon medium calcium concentration and the size of the cellular organophosphate pool. When ATP and 2,3-diphosphoglycerate contents were depleted or repleted, the cellular calcium content changed proportionally. Calcium loading of fresh erythrocytes produced no discernible change in the cellular concentrations of ATP or 2,3-diphosphoglycerate. Calcium accumulation was also accompanied by loss of cellular potassium and H₂O, deterioration of cell filterability, and spheroechinocytic transformation.     

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of ¹⁴C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.     

Regulation of lactate dehydrogenase activity in Rothia dentocariosa by fructose 1,6-diphosphate and adenosine 5''-triphosphate.

The L-(+)-lactate dehydrogenase from Rothia dentocariosa strain 17931 is activated by fructose 1,6-diphosphate and inhibited by adenosine 5'-triphosphate. The enzyme has a molecular weight of 120,000. In these respects, it resembles the lactate dehydrogenase of Actinomyces viscosus.     

Internal magnesium, 2,3-diphosphoglycerate, and the regulation of the steady-state volume of human red blood cells by the Na/K/2Cl cotransport system.

This study is concerned with the relationship between the Na/K/Cl cotransport system and the steady-state volume (MCV) of red blood cells. Cotransport rate was determined in unfractionated and density-separated red cells of different MCV from different donors to see whether cotransport differences contribute to the difference in the distribution of MCVs. Cotransport, studied in cells at their original MCVs, was determined as the bumetanide (10 microM)-sensitive 22Na efflux in the presence of ouabain (50 microM) after adjusting cellular Na (Nai) and Ki to achieve near maximal transport rates. This condition was chosen to rule out MCV-related differences in Nai and Ki that might contribute to differences in the net chemical driving force for cotransport. We found that in both unfractionated and density-separated red cells the cotransport rate was inversely correlated with MCV. MCV was correlated directly with red cell 2,3-diphosphoglycerate (DPG), whereas total red cell Mg was only slightly elevated in cells with high MCV. Thus intracellular free Mg (Mgifree) is evidently lower in red cells with high 2,3-DPG (i.e., high MCV) and vice versa. Results from flux measurements at their original MCVs, after altering Mgifree with the ionophore A23187, indicated a high Mgi sensitivity of cotransport: depletion of Mgifree inhibited and an elevation of Mgifree increased the cotransport rate. The apparent K0.5 for Mgifree was approximately 0.4 mM. Maximizing Mgifree at optimum Nai and Ki minimized the differences in cotransport rates among the different donors. It is concluded that the relative cotransport rate is regulated for cells in the steady state at their original cell volume, not by the number of copies of the cotransporter but by differences in Mgifree. The interindividual differences in Mgifree, determined primarily by differences in the 2,3-DPG content, are responsible for the differences in the relative cotransport activity that results in an inverse relationship with in vivo differences in MCV. Indirect evidence indicates that the relative cotransport rate, as indexed by Mgifree, is determined by the phosphorylated level of the cotransport system.