Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay.

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.

Membrane-bound cytochrome b5 reductase (methemoglobin reductase) in human erythrocytes. Study in normal and methemoglobinemic subjects.

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.

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes.

In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost.

Plasma membrane nadh dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species.

Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.

Platelet monamine oxidase in chronic schizophrenia. Some enzyme characteristics relevant to reduced activity.

To evaluate further the basis for the reduced activity of platelet monoamine oxidase (MAO) found in chronic schizophrenic patients, a number of characteristics of the enzyme were compared between patients and controls. Equivalent and statistically significant reductions in activity of the enzyme were found in the patients when tyramine and benzylamine were used as the substrates in comparison to previously reported reductions with tryptamine as the substrate. Michaelis constants for platelet MAO from chronic schizophrenic patients with reduced enzyme activity were not different from controls. Dialysis of the enzyme from patients and controls yielded no changes in activity. Studies of other platelet enzymes, including succinate dehydrogenase, cytochrome C reductase, and lactate dehydrogenase in patients, normal controls, and a subgroup of normal controls with reduced MAO activity, showed no parallel reductions in activity in patients or controls with low MAO activity. These findings suggest that the reduced MAO activity found in chronic schizophrenic patients is apparently not accounted for by nonspecific changes in platelets or platelet mitochondria.

Amino acid sequence of NADH-cytochrome b5 reductase of human erythrocytes.

The amino acid sequence of soluble NADH-cytochrome b5 reductase purified from normal human erythrocytes was determined as one approach to understand the hereditary disease of a deficiency of this enzyme. The protein is hydrophilic as a whole, but two regions, from Phe-36 to Ile-71 and from Met-231 to Phe-275, were found to be highly hydrophobic. The sequence of the latter region is particularly unique, and rich in proline (20%). The sequence of the amino-terminal region was very similar to the partial sequences of the corresponding regions of the enzymes from pig and steer liver microsomes.

Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes.

The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.

Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping.

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.

Purification and properties of soluble NADH-cytochrome b5 reductase of rabbit erythrocytes.

Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme was purified about 12,000-fold from hemolysate in terms of NADH-cytochrome b5 reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33,000 and 32,000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b5 reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b5 with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b5 reductase activity of the purified enzyme was very high, 1,280 mumol/min/mg of protein in 10 mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b5 were determined to be 2.5 and 4 microM, respectively. The reduction of cytochrome b5 with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20 mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed.

Exponential decay of cytochrome b5 and cytochrome b5 reductase during senescence of erythrocytes: relation to the increased methemoglobin content.

Human erythrocytes were divided into age groups according to their density using phthalate esters as separating liquids. The concentration of cytochrome b5 and the activity of NADH-cytochrome b5 reductase decreased exponentially with the age of red cells. The apparent half-life of cytochrome b5 was estimated to be 44 days. The decline of cytochrome b5 seemed to be more rapid than the decline in the activities of glutamate-oxaloacetate transaminase and NADH-cytochrome b5 reductase whose apparent half-lives were 210 and 240 days, respectively. A biphasic decline of cytochrome b5 was observed on storage of erythrocytes at 4 degrees C. It was deduced from the kinetic results that the decrease of cytochrome b5 might be involved in the increase of the concentration of methemoglobin in senescent erythrocytes. Cytochrome b5 may be used as an indicator of mean red cell age.

Antibody-based spot test for NADH-cytochrome b5 reductase activity for the laboratory diagnosis of congenital methemoglobinemia.

NADH (reduced Coenzyme I)-cytochrome b5 reductase (b5R) is a multifunctional redox enzyme, whose deficiency causes recessive congenital methemoglobinemia. A novel procedure for the detection of b5R activity in human hemolysates was developed, in which b5R monoclonal antibodies dot-blotted on nitrocellulose membrane was used to capture and enrich b5R from hemolysates, and the captured b5R activity was subsequently visualized with the substrate 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Application of this simple method to the detection of b5R activity in the hemolysates from different subjects demonstrated that it was both sensitive and reliable. Our method would be useful for the laboratory diagnosis of congenital methemoglobinemia.

Endoplasmic reticulum abnormality in Alzheimer's disease: selective alteration in platelet NADH-cytochrome c reductase activity.

Previous evidence suggests that the increase in platelet membrane fluidity associated with a subgroup of patients with Alzheimer's disease results from the accumulation of internal membrane. The specific activities of enzyme markers for selective cell membrane compartments were compared in platelets from subgroups of demented patients with normal or increased fluidity as well as from normal control subjects. A statistically significant change in enzyme activity was observed only for antimycin A-insensitive NADH-cytochrome reductase, a selective marker for smooth endoplasmic reticulum (SER) in platelets. This reduction was limited to the subgroup of demented patients who had increased platelet membrane fluidity, and therefore is not a nonspecific concomitant of neurodegeneration, medication exposure, or chronic illness in general. Since the platelet membrane alteration associated with Alzheimer's disease results from the inheritance of a single major locus, these results suggest that a defect in SER function may exist in brain cells as well as peripheral cells that express this genotype.

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label.

Electron paramagnetic resonance was used to characterize the first use of a thio-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

Methaemoglobinaemia in areas with high nitrate concentration in drinking water.

BACKGROUND: An epidemiological investigation was undertaken in all age groups to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking water. METHODS: Five areas were selected with an average nitrate concentration (as nitrate) of 26, 45, 95, 222 and 459 mg nitrate ions/litre in drinking water. These areas were visited and the house schedule (containing name, age, sex and weight of the family members) prepared in accordance with the statistically designed protocol. In all, 178 persons, matched for age and weight, were selected and arranged in five age groups. They constituted 10% of the total population of each of these areas. A detailed history of the selected population was taken, medical examination conducted and blood samples taken to ascertain the level of methaemoglobin. The collected data were subjected to statistical analysis to ascertain a relationship between nitrate concentration and methaemoglobinaemia. RESULTS: High nitrate concentrations cause severe methaemoglobinaemia (7%-27% of Hb) in all age groups, especially in the age group of less than 1 year and above 18 years. The lower levels of methaemoglobin in the age group of 1-18 years is probably due to better reserve of cytochrome b5 reductase activity and its adaptation to increasing nitrate concentration in water to compensate for methaemoglobinaemia in this age group. CONCLUSION: We conclude that high nitrate ingestion causes methaemoglobinaemia in all age groups. Cytochrome b5 reductase activity and its adaptation with increasing water nitrate ingestion plays a role in compensating for the methaemoglobinaemia.

Recurrent diarrhea in children living in areas with high levels of nitrate in drinking water.

Given that there was documented evidence of an association between diarrhea and high nitrate ingestion, the authors examined drinking water nitrate concentration and its possible correlation(s) with methemoglobin levels, cytochrome b5 reductase activity, and recurrent diarrhea. In addition, the authors studied histopathological changes in the intestines of rabbits in an animal model. Five village areas were studied, and nitrate concentrations (expressed in mg of nitrate per liter of water) of 26, 45, 95, 220, and 459 existed in the respective villages. The study included 88 randomly selected children who were 8 yr of age or younger; they represented 10% of the total population of each of the areas. Detailed histories of recurrent diarrhea were noted, and medical examinations were conducted. Cytochrome b5 reductase activity and methemoglobin levels were estimated biochemically. Collected data were analyzed statistically with Microsoft Excel software. In addition, the authors exposed rabbits to various levels of nitrate, and histopathological changes of the stomach and intestine (small and large) were evaluated. There was a strong relationship between nitrate concentration and recurrent diarrhea; 80% of the recurrent diarrhea cases were explained by nitrate concentration alone. In the rabbit intestines, lymphocytic infiltration and hyperplasia characterized the submucosa as nitrate concentrations increased.