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Membrane-Bound Cytochrome B5 Reductase (Methemoglobin Reductase) in Human Erythrocytes

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Membrane-Bound Cytochrome B5 Reductase (Methemoglobin Reductase) in Human Erythrocytes Membrane-bound cytochrome b5 reductase (methemoglobin reductase) in human erythrocytes. Study in normal and methemoglobinemic subjects. D Choury, … , A Leroux, J C Kaplan J Clin Invest. 1981;67(1):149-155. https://doi.org/10.1172/JCI110007. Research Article In this study we present evidence that in human erythrocytes NADH-cytochrome b5 reductase (methemoglobin reductase) is not only soluble but also tightly bound to the membrane. The membrane methemoglobin reductase-like activity is unmasked by Triton X-100 treatment, and represents about half of the total activity in the erythrocytes. Like the amphiphilic microsomal-bound cytochrome b5 reductase, the erythrocyte membrane-bound enzyme is solubilized by cathepsin D. Because this treatment is effective on unsealed ghosts but not on resealed (inside-in) ghosts, it is concluded that the enzyme is strongly bound to the inner face of the membrane. The erythrocyte membrane enzyme is antigenically similar to the soluble enzyme. The two forms of enzyme are specified by the same gene, in that both were found defective in six patients with recessive congenital methemoglobinemia. We suggest that the cytochrome b5 reductase of the erythrocyte membrane is the primary gene product. A posttranslational partial proteolysis probably gives rise to the soluble form of the enzyme, which serves as a methemoglobin reductase. Find the latest version: https://jci.me/110007/pdf Membrane-bound Cytochrome b5 Reductase (Methemoglobin Reductase) in Human Erythrocytes STUDY IN NORMAL AND METHEMOGLOBINEMIC SUBJECTS DANItLE CHOURY, ALENA LEROUX, and JEAN-CLAUDE KAPLAN, Institut de Pathologie Moleculaire, Institut National de la Sante et de la Recherche Medicale U 129, 75014 Paris, France A B S T R A C T In this study we present evidence that actually a soluble NADH-cytochrome b5 reductase in human erythrocytes NADH-cytochrome b5 reduc- (5-8) that reduces a soluble cytochrome b5. The re- tase (methemoglobin reductase) is not only soluble but duced cytochrome b5 interacts directly with methemo- also tightly bound to the membrane. The membrane globin (5, 9). In the other cells, the NADH-cytochrome methemoglobin reductase-like activity is unmasked by b5 reductase is predominantly bound to the mem- Triton X-100 treatment, and represents about halfofthe branes: endoplasmic reticulum (10) and mitochondria total activity in the erythrocytes. Like the amphiphilic (11). However, a soluble form of the enzyme has also microsomal-bound cytochrome b5 reductase, the eryth- been found in the cytosolic fraction of human placenta rocyte membrane-bound enzyme is solubilized by (8) and rabbit liver (12). cathepsin D. Because this treatment is effective on In this paper, we report the finding of a NADH- unsealed ghosts but not on resealed (inside-in) ghosts, cytochrome b5 reductase that is strongly attached to it is concluded that the enzyme is strongly bound to the the inner face of the erythrocyte membrane and is re- inner face of the membrane. The erythrocyte mem- leased either by detergent treatment or by partial brane enzyme is antigenically similar to the soluble digestion by cathepsin D. The immunologic and enzyme. The two forms of enzyme are specified by the genetic characterization of the membrane-bound en- same gene, in that both were found defective in six zyme suggests that it is produced by the same gene patients with recessive congenital methemoglobinemia. (DIA1) as the soluble erythrocyte diaphorase. We suggest that the cytochrome b5 reductase of the erythrocyte membrane is the primary gene product. A METHODS posttranslational partial proteolysis probably gives rise to the soluble form of the enzyme, which serves as a Preparation of the erythrocyte membranes methemoglobin reductase. The erythrocyte membranes were prepared according to Marchesi et al. (13). Washed human erythrocytes were INTRODUCTION hemolysed by 30 vol of 5 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, and the 22,000-g pellet was extensively washed in The so-called methemoglobin-reductase or NADH- the same medium to obtain a completely white membrane diaphorase has been described long ago as a soluble preparation. For the preparation of inside-in resealed ghosts, we used the procedure of Steck and Kant (14) in which erythrocyte enzyme that has a major role in the enzy- hemolysis is initiated by mixing 1 vol of washed erythrocytes matic reduction of methemoglobin (1). An inherited with 40 vol of 5 mM sodium phosphate buffer, pH 8, containing homozygous defect of this enzyme produces congenital 1 mM MgSO4. recessive methemoglobinemia (2). The enzyme locus (DIA,) has been assigned to chromosome 22 (3, 4). It Extraction has been demonstrated that this erythrocyte enzyme is With detergent. The ghosts were suspended in a 10 mM Tris-HCl buffer, pH 7.4, containing 2% Triton X-100. After Preliminary data have been presented at the Twenty- 30 min of incubation at 4°C, the suspension was frozen and second annual meeting ofthe American Society of Hematology thawed three times and centrifuged for 10 min at 105,000 g, in 1979. Blood. 541 (Suppl. 1): 342. (Abstr.) at 4°C, in a Beckman Airfuge (Beckman Instruments Inc., Received for publication 14 July 1980 and in revised form Fullerton, Calif.) centrifuge to spin down small volumes 15 September 1980. (170 1l). The supematant was assayed for enzyme activity. J. Clin. Invest. (D The American Society for Clinical Investigation, Inc. - 0021-9738181/01/0149/07 $1.00 149 Volume 67 January 1981 149-155 TABLE I of Ellman et al. (18); glyceraldehyde-3-phosphate dehydroge- Effect of Triton X-100 on Erythrocyte nase was assayed according to Beutler et al. (19). Methemoglobin Reductase Proteins were estimated by the method of Lowry et al. When large amount of Triton X-100 produced a precipitate in the Without With final mixture, it was discarded by 5-min centrifugation at Triton X- 100 Triton X-100 10,000 rpm. Crude unspun hemolysate, nmollmin/mg Hb 2.5±0.5 4.8+0.6 Electrophoresis membranes, Washed erythrocyte Horizontal starch gel electrophoresis was performed as nmollminlrng protein 3.0+0.5 78+±10 described by Kaplan and Beutler (20) using a Tris-EDTA borate buffer, pH 8.6. Polyacrylamide gel isoelectrofocusing In both cases the enzyme was assayed according to Hegesh was carried out in a pH 6-8 linear gradient (Ampholines; et al. (15). The detergent-treated hemolysate and the detergent- LKB Instruments, Inc., Orsay, France) according to the treated membranes were incubated 30 min at 4°C with 2% method of Drysdale et al. (21). In all the electrophoretic Triton X-100 (final concentration) before the assay. studies specific staining for NADH-diaphorase activity was performed with a mixture containing 1.2 mM NADH, 0.06 mM dichlorophenol indophenol, and 1.2 mM MTT in a 0.25 With cathepsin D. A 1:1 suspension of membranes in M Tris-HCl buffer (pH 8.4) (20). 0.1 M Tris-maleate, pH 5.6, was incubated for 2 h at 37°C with cathepsin D (5 ,ug/10 mg membrane protein). The prepa- ration was then centrifuged at 105,000 g as described above. Imniunological studies The supernatant was assayed for enzyme activity. Inlactivation by antiserum. The different preparations were incubated with a chicken antiserum prepared against hemolysate human erythrocyte-soluble cytochrome b5 reductase (8) in the Preparation of membrane-free presence of 5% (vol/vol) of polyethylene glycol. Increasing Washed erythrocytes were hemolysed with 4 vol of bidis- amounts of antiserum were added to constant amounts (ex- tilled water and centrifuged at 105,000 g for 10 min at 4°C in pressed as units of methemoglobin-ferrocyanide reducing the Beckman Airfuge centrifuge. The supernatant contained activity) of enzyme. After incubation for 1 h at 40C followed the soluble enzyme. by centrifugation (20 min at 22,000 g) the residual activity was measured in the supernatant. Double irmmunodiffusion. Double immunodiffusion was Preparation of semipurified soluble performed according to Ouchterlony (22). Antigens and anti- cytochrome b5 reductase from serum were incubated for 2-3 d and then the plate was ex- tensively washed with isotonic saline solution for 2 d to re- human erythrocytes move the excess enzyme that had not reacted with the anti- The membrane-free hemolysate was diluted with 2 vol of serum. The precipitation lines were specifically visualized a 5 mM potassium phosphate buffer, pH 7.0, anid adjusted to usinig the NADH-diaphorase staining method (20). the same pH. Hemoglobin was removed from the diluted hemolysate by a DEAE-cellulose 52 batchwise treatment in the same buffer using 2 g of preswollen resin/ml of packed Chemicals erythrocytes. The resin was extensively washed in a Bucher funnel with the buffer unitil the effluent was colorless. The NADH, 2,6-dichlorophenol indophenol, MTT, Triton enzyme was eluted with 50 mM potassium phosphate buffer, X-100, potassium ferricyanide, cathepsin D, D-L glyceralde- 0.1 mM EDTA and 0.3 NI KCI, and pre- hyde-3-phosphate, adenosine 5-triphosphate, 5,5 dithiobis- pH 5.8 that contained (2 nitrobenzoic acid), acetyl thiocholine iodide were from cipitated by (NH4)2SO4 added to 60% saturationi. The precipi- DEAE-cellulose 52 was tate was separated by centrifugation and dialyzed against a Sigma Chemical Co., St. Louis, Mo. Tris-HCl buffer 10 mM, pH 7.5, overnight at 4°C f'or enzymle from \Vhatman Inc., Clifton, N. J. assays. This procedure yielded a hemoglobin-free preparation of the soluble cytochrome b, reductase. RESULTS E)izymJe E rythtrocyte memtbran e methemiooglobin reductase assayjs aictivity. Treating a noncentrifuged 1:4 hemolysate All assays were carried out at 25°C. The NADH-imiethenmo- by 2% Triton X-100 (final concentration) doubles the globin reductase activity was assayed with ferrocyanide methemoglobin complex as an acceptor, according to Hegesh activity of the enzyme assayed by the method of et al.
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