NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme

A NADPH-dehydrogenase of human erythrocytes was exhaustively purified to a homogeneous protein judging from the electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. Studies on the specificity for the electron acceptor of this enzyme suggest that flavins serve as the natural and direct electron acceptor. The enzyme showed a broad specificty for flavins and the Michaelis constants for flavins were estimated to be 5 × 10^?5 M for both FMN and riboflavin. Rapid reduction of methemoglobin by the enzyme in the presence of flavin was demonstrated, and the reduction was explained by the reduction of flavin by the enzyme, and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin. The therapeutic significance of flavins was discussed with reference to the flavin reductase activity in hereditary methemoglobinemia.     

Characterization of glutathione reductase from porcine erythrocytes.

Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) was purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2',5'-ADP-Sepharose 4-B. Analytical ultracentrifugation experiments were analysed to give the following physical parameters for the enzyme: s20,w = 5.7 S, D20,w = 50 microgram2/s, and Mw = 103 000 (protein concentration, 0.5 mg/ml). The frictional ratio was 1.37 and the Stokes radius was 4.3 nm. The enzyme molecule is a dimer composed of subunits of equal size each containing a FAD molecule. The amino acid compositions and circular dichroism spectra of the porcine and human enzymes indicated extensive structural similarities. The isoelectric point was at pH 6.85 (at 4 degrees C). The absorption spectrum of the oxidized enzyme had maxima at 377 and 462 nm. In vivo the enzyme appears to be partially reduced. At a physiological concentration of reduced glutathione the apparent Michaelis constants for glutathione disulfide and NADPH were higher than in the absence of reduced glutathione. At 0.15 M ionic strength the catalytic activity obtained with NADPH as reductant was optimal at pH 7 and more than 200 times higher than that obtained with NADH. S-sulfoglutathione and some mixed disulfides of glutathione were poor substrates with the exception of the mixed disulfide of coenzyme A and reduced glutathione. The purified enzyme displayed low transhydrogenase activity with oxidized pyridine nucleotide analogs and diaphorase activity with 2,6-dichlorophenolindophenol as acceptor substrates; both NADPH and NADH served as donors.     

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.     

Changes in intermediate haemoglobins during methaemoglobin reduction by NADPH-flavin reductase.

The changes in intermediate haemoglobins produced during methaemoglobin reduction by NADPH-flavin reductase were analysed by an isoelectric-focusing method. The alpha 3+ beta 2+ and alpha 2+ beta 3+ valency hybrids were observed as intermediate haemoglobins and changed consecutively with time during the reaction. On the basis of the analyses, the course of methaemoglobin reduction was found to involve two different pathways: (1) methaemoglobin kappa+1 leads to alpha 3+ beta 2+ kappa+2 leads to oxyhaemoglobin; (2) methaemoglobin kappa+3 leads to alpha 2+ beta 3+ kappa+4 leads to oxyhaemoglobin. The reaction rate constants of each phase (kappa+1--kappa+4) were also estimated. The addition of inositol hexaphosphate to the reaction mixture did not affect the overall reaction. The mechanism of methaemoglobin reduction by NADPH-flavin reductase is discussed on the basis of these results.     

Glutathione reductase from human erythrocytes. Catalytic properties and aggregation.

The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides optimal conditions for determining the basic kinetic parameters of the enzyme. The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates. In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site. The relation of the properties of glutathione reductase to cellular conditions is discussed.     

Characterization of PEG-mediated electrofusion of human erythrocytes.

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5'-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.     

Soluble and microsomal forms of NADH-cytochrome beta 5 reductase from human placenta. Similarity with NADH-methemoglobin reductase from human erythrocytes.

In congenital methemoglobinemia associated with mental retardation a generalized deficiency of NADH-cytochrome beta 5 reductase (NADH : ferricytochrome beta 5 oxidoreductase, EC 1.6.2.2) has been found in soluble extracts of red blood cells, as well as in deoxycholate-treated extracts of leukocytes, muscle, liver and fibroblasts (Leroux et al. (1975) Nature 258, 619-620). In the present study the relationship between the microsomal (I) and the soluble (II) NADH-cytochrome beta 5 reductase was investigated, using human placenta as a source of enzyme. Both forms were compared to the human red-cell soluble NADH-methemoglobin reductase (III) and NADH-cytochrome beta 5 reductase (IV). The four entities exhibited great immunological similarities. It is concluded that the three soluble enzymes (II, III and IV) are identical. The detergent-solubilized microsomal NADH-cytochrome beta 5 reductase (I) is immunologically very similar to the soluble enzymes, but presents distinct features possibly due to the presence of a hydrophobic part.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Characterization of the glucose transporter from human erythrocytes

The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394–7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight. The K_D (0.14 μM) and extent (11 nmoles/mg protein) for binding of ³H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.