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Concentration of NADH-Cytochrome B5 Reductase in Erythrocytes of Normal and Methemoglobinemic Individuals Measured with a Quantitative Radioimmunoblotting Assay

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Concentration of NADH-Cytochrome B5 Reductase in Erythrocytes of Normal and Methemoglobinemic Individuals Measured with a Quantitative Radioimmunoblotting Assay Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay. N Borgese, … , G Pietrini, S Gaetani J Clin Invest. 1987;80(5):1296-1302. https://doi.org/10.1172/JCI113205. Research Article The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity. Find the latest version: https://jci.me/113205/pdf Concentration of NADH-Cytochrome b5 Reductase in Erythrocytes of Normal and Methemoglobinemic Individuals Measured with a Quantitative Radioimmunoblotting Assay Nica Borgese, Grazia Pietrini, and Sancia Gaetani* Consiglio Nazionale delle Ricerche Center of Cytopharmacology, 20129 Milan, Italy; and *Istituto Nazionale della Nutrizione, 00100 Rome, Italy Abstract retardation, both the erythrocyte soluble protein and the membrane-bound forms of other tissues are lacking (1 1); The activity of NADH-cytochrome b5 reductase (NADH-met- therefore, it is thought that the two forms of the enzyme are hemoglobin reductase) is generally reduced in red cells of pa- products of the same gene (see 12 for a review). In most cases tients with recessive hereditary methemoglobinemia. To deter- of hereditary enzymatic methemoglobinemia, however, the mine whether this lower activity is due to reduced concentra- levels of reductase are selectively depressed in erythrocytes and tion of an enzyme with normal catalytic properties or to are normal, or close to normal (1 1, 13), in other cell types; in reduced activity of an enzyme present at normal concentration, these cases, the disease is mild and not associated with neuro- we measured erythrocyte reductase concentrations with a logical disorders. Recently, a second type of mild disease has quantitative radioimmunoblotting method, using affinity-puri- been reported in which the enzyme is deficient in both red and fied polyclonal antibodies against rat liver microsomal reduc- white blood cells and presumably present at normal levels in tase as probe. In five patients with the "mild" form of recessive other tissues ( 14). Destruction of an altered enzyme by proteo- hereditary methemoglobinemia, in which the activity of eryth- lytic enzymes present in blood cells and not in other tissues rocyte reductase was 4-13% of controls, concentrations of the could explain this selective deficit of the reductase in the eryth- enzyme, measured as antigen, were also reduced to 7-20% of rocytes (and in some cases leukocytes) of patients with the the control values. The concentration of membrane-bound re- mild form of the disease (15). However, until the present, only ductase antigen, measured in the ghost fraction, was similarly measurements of the enzyme activity of methemoglobinemic reduced. Thus, in these patients, the reductase deficit is caused patients have been done, whereas data on the levels of reduc- mainly by a reduction in NADH-cytochrome b5 reductase tase protein are not available. Thus, it is possible that methe- concentration, although altered catalytic properties of the en- moglobinemic individuals have normal levels of an enzyme zyme may also contribute to the reduced enzyme activity. with reduced activity. In this study, we have undertaken to directly measure levels of cyt b5 reductase in erythrocytes of Introduction methemoglobinemic patients and normal subjects with a quantitative radioimmunoblotting assay, using an affinity-pu- The principal enzymatic system of the red cell for the reduc- rified polyclonal antibody against the rat liver microsomal en- tion of methemoglobin (MetHb)' consists of a soluble zyme as probe. NADH-cytochrome b5 (cyt b5) reductase (1) and an interme- diate electron carrier, soluble cyt b5 (2). Soluble erythrocyte Methods reductase is very similar enzymatically (1) and immunologi- cally (3, 4) to a membrane-bound form of the enzyme, present Materials. In addition to materials listed in previous publications (16, on endoplasmic reticulum and outer mitochondrial mem- 17), the following chemicals were purchased from the indicated branes of a variety of tissues. Sequencing studies of the human sources: a-cellulose and Sigmacell type 50. Sigma Chemical Co., St. erythrocyte (5) and steer liver microsomal (6) reductases have Louis, MO; 5'AMP-Sepharose 4B, Biotechnology International AB, confirmed the high degree of homology between the two forms Uppsala, Sweden; hemoglobin assay kit, Boehringer Mannheim of the enzyme, showing also that the soluble form lacks a GmbH, Mannheim, FRG.; 50% glutaraldehyde, Fluka AG, Buchs, hydrophobic segment at the NH2-terminus, present in the mi- Switzerland. crosomal reductase. Purification ofproteins and preparation ofantibodies. The purifica- tion of the water-soluble fragment of rat liver microsomal NADH-cyt In hereditary recessive methemoglobinemia, the activity of b5 reductase, the production of antisera in rabbits, and the purification erythrocyte cyt b5 reductase is generally reduced (7-9, but also of antireductase antibodies by affinity chromatography have been de- see 10). In a rare form of the disease, associated with mental scribed in previous publications (16, 18). Briefly, the Cathepsin D-sol- ubilized form of cyt b5 reductase, purified by standard procedures and Address reprint requests to Dr. Borgese, C.N.R. Center of Cytophar- conjugated to keyhole limpet hemocyanin, was used as antigen. Anti- mocology, via Vanvitelli 32, 20129 Milan, Italy. sera were passed over a column containing Sepharose B conjugated to Receivedfor publication 19 May 1986 and in revisedform 2 April the same reductase preparation as the one used as antigen. Antireduc- 1987. tase antibodies were eluted with a pH 2.2 buffer. The resulting affin- ity-purified antibodies recognize only one band on immunoblots of rat 1. Abbreviations used in this paper: cyt b5, cytochrome b5; MetHb, liver microsomes and human erythrocytes (see Fig. 2), and immuno- methemoglobin; MetHb.FeCN, MetHb-ferrocyanide complex. precipitate a polypeptide that yields two-dimensional peptide maps indistinguishable from those obtained with the enzyme purified by J. Clin. Invest. standard procedures ( 18). © The American Society for Clinical Investigation, Inc. A partially purified preparation of human erythrocyte soluble cyt 0021-9738/87/11/1296/07 $2.00 b5 reductase was obtained from 225 ml of packed red blood cells. Cells Volume 80, November 1987, 1296-1302 were lysed with 36 vol of hypotonic buffer (1 mM EDTA, 1 mM 1296 N. Borgese, G. Pietrini, and S. Gaetani phenylmethylsulfonyl fluoride (PMSF), and 5 mM Na+ phosphate were removed from the rat blood by passage through the microcrystal- buffer, pH 7), and centrifuged at 25,000 g. for 30 min (Sorvall GSA line cellulose-a-cellulose column. rotor, DuPont de Nemours & Co., Inc., Sorvall Instruments Div., Washed erythrocytes were lysed, and the lysate fractionated into a Newton, CT). Soluble reductase was recovered in the resulting super- ghost and soluble fraction, as previously described (16), but the last nate, which was supplemented with solid (NH4)2SO4 to a final concen- wash of the ghost pellet was with PBS. Before SDS-PAGE, the soluble tration of 64.5% of saturation. After allowing the (NH4)2SO4 to dis- fraction was concentrated by liophylization followed by resuspension solve, the suspension was left for 2 h at 4VC and then centrifuged at in one-tenth the original volume of PBS. In one experiment the soluble 25,000 g. for 10 min (Sorvall GSA rotor, DuPont de Nemours & reductase was concentrated by (NH4)2504 precipitation (16). Co., Inc.). The supernate was discarded, and the precipitate was taken Biochemical assays. Reductase activity in whole blood or erythro- up with phosphate buffered saline (PBS) containing 1 mM EDTA plus cyte preparations was determined by the rate of reduction of the ferri- I mM PMSF to a volume of 150 ml. This solution was concentrated to hemoglobin-ferrocyanide complex (Methb.FeCN), as described by 25 ml by ultrafiltration (Amicon PM-10 filter; Amicon Corp., Hegesh et al. (23) with slight modification. The hemoglobin (Hb) sub- Danvers, MA), and dialysed extensively against PBS plus I mM EDTA strate preparation was centrifuged before use (40,000 gma. for 30 min plus I mM PMSF. After addition of Triton X-100 and EDTA to final [Sorvall SA600 rotor; DuPont de Nemours & Co., Inc.]), and addi- concentrations of 2% and 5 mM, respectively, the solution was clari- tional centrifugation after addition of the sample was found to be fied by ultracentrifugation and then loaded
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