Phosphorus compounds in the human erythrocyte

Bartlett, Grant R.

doi:10.1016/0304-4165(68)90251-1

Cited by 81 publications

(14 citation statements)

References 17 publications

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“…2 were determined enzymatically with yeast hexokinase; since hexokinase can react with other nucleoside triphosphates (15), it was necessary to verify the identity of the ATP. Recovered erythrocytes, at day 51, were briefly incubated with [32P]phosphate, followed by perchloric acid precipitation and Dowex-1 chromatography by standard techniques (13). The chromatographic profiles shown in Fig.…”

Section: Figurementioning

confidence: 99%

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Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.

Suzuki

Dale

1988

Proc. Natl. Acad. Sci. U.S.A.

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Rabbit erythrocytes were covalently labeled with biotin and then infused into the donor animial. Up to 60 days after infusion, the biotinylated cells were selectively isolated by their affinity for avidin. These aged erythrocytes, which are within 10 days of death, were analyzed for several biochemical parameters. The 2,3-bisphosphoglycerate and glutathione levels of these cells were constant with age; however, the adenosine 5'-triphosphate concentrations increased approximately 75% as the cells approached the end of their life span. The activities of several glycolytic enzymes did not change with age.The mechanisms of erythrocyte senescence are poorly understood (1). Clearly, erythrocytes have a well-defined life span; however, the study of the aging processes in these cells has been hindered by the inability to definitively isolate senescent erythrocytes. A myriad of publications have reported age-related differences in various biochemical parameters of erythrocytes (for a review, see ref. 1). However, the majority of these are based on the separation of cells over density gradients with the underlying assumption that the most dense fractions represent the old cells, an assumption that has been questioned (2,3).Two methods that are independent of the physical separation techniques outlined above have been reported for the isolation of senescent erythroctyes. Allison and Burn (4) transfused blood group 0 erythrocytes into normal blood group A individuals. At subsequent time points, the 0 cells were recovered after agglutination of the host A erythrocytes. Unfortunately, the technique was hindered by technical difficulties. More recently, Ganzoni et al. (5) Preparation of Young Erythrocyte Cohorts. In some experiments, young erythrocyte populations were used for biotinylation and replacement into donor rabbits. The advantage of using a young cohort of cells is that a larger percentage of the cells will still be circulating 50-60 days after replacement and therefore will allow recovery of greater numbers of senescent cells. Two procedures were used to produce young cell populations-phenylhydrazine treatment and repeated phlebotomy. Phenylhydrazine hydrochloride (25 mg) was subcutaneously injected into a rabbit on days 0, 2, and 3. On day 13, 50 ml of blood was collected in heparin for biotinylation. The reticulocyte count for these preparations averaged more than 50% on day 8. For the repeated phlebotomy procedure, 50-60 ml of blood was drawn from the rabbit on day 0, 2, and 4. The sample for biotinylation was taken on day 14. To protect against iron deficiency during repeated phlebotomy, each rabbit received 50 mg of iron-dextran intramuscularly on days -7, -5, -3, 0, 2, and 4. Preparation and Infusion of Biotinylated Erythrocytes.Rabbit erythrocytes were washed, labeled with ['4C]cyanate, treated with NHS-biotin, and infused into the donor rabbit exactly as described (7). The average level of biotinylation was approximately 25,000 biotin molecules per erythrocyte.Avidin-Coated Polystyrene Beads. The av...

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Section: Figurementioning

confidence: 99%

“…Precipitated protein was spun down, and the perchloric acid supernatant was neutralized as detailed above. The extract was chromatographed as detailed (13).…”

Section: Preparation and Infusion Of Biotinylated Erythrocytesmentioning

confidence: 99%

Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.

Suzuki

Dale

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…3). In normal cells that are pulse labeled with 32P4O, the activity of the f,-and -y-phosphate thus quickly becomes equal (15). In our studies, labeling was allowed to continue, so that the extent of labeling of the y-position was somewhat higher than that of the f#-phosphate, even in the normal control.…”

Section: Resultsmentioning

confidence: 78%

Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

Beutler¹,

Carson²,

Dannawi³

et al. 1983

J. Clin. Invest.

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A B S T R A C T A child with hemolytic anemia was found to have severe erythrocyte adenylate kinase (AK) deficiency, but an equally enzyme-deficient sibling had no evidence of hemolysis. No residual enzyme activity was found in erythrocytes by spectrophotometric methods that could easily have detected 0.1% of normal activity. However, concentrated hemolysates were shown to have the capacity to generate small amounts of ATP and AMP from ADP after prolonged incubation. Hemolysates could also catalyze the transfer of labeled -y-phosphate from ATP to ADP. Intact erythrocytes were able to transfer phosphate from the "y-position of ATP to the ,B-position, albeit at a rate substantially slower than normal. They could also incorporate 4CG-labeled adenine into ADP and ATP. Thus, a small amount of residual AK-like activity representing about 1/2,000 of the activity normally present could be documented in the deficient erythrocytes. The residual activity was not inhibited by N-ethylmaleimide, which completely abolishes the activity of the normal AK1 isozyme of erythrocytes. The minute amount of residual activity in erythrocytes could represent a small amount of the AK2 isozyme, which has not been thought to be present in erythrocytes, or the activity of erythrocyte guanylate kinase with AMP substituting as substrate for GMP.Peripheral blood leukocytes, cultured skin fibroblasts, and transformed lymphoblasts from the deficient subject manifested about 17, 24, and 74%, respectively, of the activity of the concurrent controls. This residual activity is consistent with the existence of genetically independent AK isozyme, AK2, which is known to exist in these tissues.

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“…The DPG contents in the control REDUCTION OF CANINE BLOOD 02-AFFINITY 535 affinity blood were measured enzymatically (MAEDA et al, 1971). Isolations and quantitative characterizations of acid soluble phosphate compounds in the red cells were performed by the anion-exchange chromatographic method of BARTLETT (1968) as. modified by OCHIAI and ENOKI (1975).…”

Section: Methodsmentioning

confidence: 99%

Incubation procedure effective for decreasing oxygen affinity of red cells in the dog.

et al. 1986

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Because oxygen affinity of canine red cells had been hardly reduced by the conventional procedure of incubation with inosinepyruvate-phosphate or dihydroxyacetone-pyruvate-phosphate, incubation with acidified phosphoenolpyruvate (PEP) medium was tested for its effect on oxygen affinity. As this procedure had been shown to induce a significant increase of 2,3-diphosphoglycerate (DPG) in human red cells, it was also proved to be effective for lowering oxygen affinity of canine red cells. Incubation with 15 mM PEP in ACD medium (pH 6.0) at 37°C for 30 min was the standard procedure established after critical scrutiny. The incubated blood exhibited an average increase in half-saturation pressure (P50) amounting to 7 Torr (pH 7.40, P~02 40 Torr, 37°C), with no appreciable hemolysis and little change in mean corpuscular hemoglobin concentration. Results with Dowex 1-X8 anion exchange chromatography of acid soluble phosphates from the incubated red cells suggested that PEP penetrated rapidly through, the canine red cell membrane and was metabolized to DPG via the retrograde process of the Embden-Meyerhof glycolytic pathway.

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Phosphorus compounds in the human erythrocyte

Cited by 81 publications

References 17 publications

Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.

Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.

Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

Incubation procedure effective for decreasing oxygen affinity of red cells in the dog.

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