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      Hemoglobin Content Is Increased in the Human Erythroleukemia K562 Cell Line by a 56.2-kD Peptide from Uremic Plasma

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          Background: Some abnormalities in porphyrin metabolism have been described in erythrocytes from patients with end-stage renal failure. A peptidic fraction of 56.2 kD isolated from plasma of these patients was previously identified as an aminolevulinate dehydratase inhibitor. The aim of this study was to examine the in vitro effect of this peptide on heme synthesis in the erythroleukemia K562 cells. Methods: The 56.2-kD fraction was purified from uremic plasma by protein electroelution and, its action on the mitochondrial rate-limiting steps of heme synthesis, as well as the hemoglobin content during erythroid differentiation induced by sodium butyrate, was investigated in K562 cells. Results: Two hours after addition of the 56.2-kD peptide, the activities of aminolevulinate acid synthase and aminolevulinate dehydratase were reduced while the activity of the ferrochelatase was enhanced, indicating that this peptide easily across the membranes. A 3-day incubation with this peptide enhanced approximately twofold the hemoglobin and porphyrin levels during erythroid differentiation of K562 cells without variation of cell growth. Conclusion: This study shows that the addition of the 56.2-kD uremic factor to K562 cells was clearly implicated in heme disturbances existing in chronic renal failure but it did not play a negative role in the pathogenesis of the uremic anemia.

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          Control of hemoglobin synthesis in erythroid differentiating K562 cells. I. Role of iron in erythroid cell heme synthesis.

          K562 cells were used to investigate the factors that control hemoglobin (Hb) synthesis. Treatment with sodium butyrate enhanced Hb synthesis and glycophorin A expression. delta-Aminolevulinate synthase (ALAS) activity and Hb levels simultaneously increased to a similar extent and with a similar time course, and the increases were dependent on the concentration of diferric transferrin (FeTf) in the culture medium. Addition of exogenous delta-aminolevulinic acid (ALA) resulted in a dose-dependent increase in Hb content. Hb synthesis was inhibited 50% after addition of succinylacetone (SA), a potent inhibitor of delta-aminolevulinate dehydratase. These findings suggest that ALAS is a key enzyme in the eight steps of de novo heme synthesis and that iron, including FeTf, plays a central role in Hb synthesis through control of ALAS activity in erythroid differentiating cells. On the other hand, erythropoietin (EPO) treatment had no effect on Hb synthesis and slightly suppressed glycophorin A expression. Hemin enhanced Hb synthesis in the K562 cells but not glycophorin A expression. The addition of ALA, SA, or FeTf to hemin-treated cells caused no significant changes in Hb synthesis. Butyrate, EPO, and hemin acted on the K562 cells in different ways and caused different biochemical changes in the Hb synthesis process.
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            Inhibition of Erythrocyte Aminolevulinate Dehydratase by a 56.2-kD Peptide from Uremic Plasma

            Among the abnormalities in erythrocyte porphyrin metabolism already described in patients with chronic renal failure on hemodialysis, a decrease in blood aminolevulinate dehydratase activity has been reported, suggesting the presence in uremic plasma of an inhibitor of the enzyme. The aim of this work has been to isolate and characterize such an inhibitor. Blood samples from 105 patients with chronic uremia were collected; plasma was applied to Sephadex G-100 columns and the fraction with the highest inhibiting capacity was identified and purified by subsequent SDS-polyacrylamide gel electrophoresis, followed by electroelution and electroblotting. It was demonstrated that the factor present in plasma of uremic patients inhibited blood aminolevulinate dehydratase in a concentration-dependent manner; its inhibitory properties were abolished after heat, trypsin and TCA treatment indicating its peptidic nature. The purified inhibitor has an apparent molecular mass of 56.2 kD, it inhibits blood aminolevulinate dehydratase in a competitive way and the K i value is 12×10 –6 M . The amino acid composition of the inhibitor has been determined and it has been found that its N-terminal amino acid is blocked. The isolated peptide may play a role in heme biosynthesis disturbances and in the pathogenesis of uremic anemia.

              Author and article information

              Am J Nephrol
              American Journal of Nephrology
              S. Karger AG
              April 2004
              08 April 2004
              : 24
              : 2
              : 230-234
              aCentro de Investigación, Hospital 12 de Octubre, Madrid, Spain; bCentro de Investigaciones sobre Porfirinas y Porfirias, CIPYP, CONICET-UBA i cInstituto Argentino de Riñón y Transplante, Buenos Aires, Argentina
              77345 Am J Nephrol 2004;24:230–234
              © 2004 S. Karger AG, Basel

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              Tables: 2, References: 17, Pages: 5
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              Original Report: Laboratory Investigation


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