Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc₁ isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc₁ is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc₁ was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc₁ retained nearly full activity, absorption spectra, subunit, and phospholipid composition.     

Purification of bovine liver microsomal NADH-cytochrome b5 reductase using affinity chromatography

Microsomal NADH-cytochrome $\text{b}{}_5$ reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × $\text{g}$ microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome $\text{b}{}_5$ is also obtained.     

Isolation of the light-harvesting chlorophyll a/b--protein complex from thylakoid membranes of barley by adsorption chromatography on controlled-pore glass.

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll $\text{a}\text{b}$-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.     

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.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Electrostatic properties deduced from refined structures of NADH-cytochrome b5 reductase and the other flavin-dependent reductases: Pyridine nucleotide-binding and interaction with an electron-transfer partner

Electrostatic properties on the protein surface were examined on the basis of the crystal structure of NADH-cytochrome b₅ reductase refined to a crystallographic R factor of 0.223 at 2.1 Å resolution and of the other three flavin-dependent reductases. A structural comparison of NADH-cytochrome b₅ reductase with the other flavin-dependent reductases, ferredoxin-NADP⁺ reductase, phthalate dioxygenase reductase, and nitrate reductase, showed that the α/β structure is the common motif for binding pyridine nucleotide. Although the amino acid residues associated with pyridine nucleotide-binding are not conserved, the electrostatic properties and the location of the pyridine nucleotide-binding pockets of NADH-requiring reductases were similar to each other. The electrostatic potential of the surface near the flavin-protruding side (dimethylbenzene end of the flavin ring) of NADH-cytochrome b₅ reductase was positive over a wide area while that of the surface near the heme-binding site of cytochrome b₅ was negative. This implied that the flavin-protruding side of NADH-cytochrome b₅ reductase is suitable for interacting with its electron-transfer partner, cytochrome b₅. This positive potential area is conserved among four flavin-dependent reductases. A comparison of the electron-transfer partners of four flavin-dependent reductases showed that there are significant differences in the distribution of electrostatic potential between inter-molecular and inter-domain electron-transfer reactions. © 1996 Wiley-Liss, Inc.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

Summary An NADH-cytochromeb ₅ reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb ₅ reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb ₅ reductases.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - BCA bicinchonicic acid - EDTA ethylenediamine tetraacetate acid disodium salt - FeCN ferricyanide - HPLC high-performance liquid chromatography - NADH nicotinamide adenine dinucleotide reduced form - PMSF -phenylmethylsulfonyl fluoride     

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450 -dependent enzyme activities in liver microsomes

The role of NADH-cytochrome b₅ reductase and cytochrome b₅ as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b₅. Antibody prepared against NADH-cytochrome b₅ reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b₅ and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b₅ in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b₅ reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b₅ to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b₅ involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b₅ functions as an intermediate electron carrier between NADH-cytochrome b₅ reductase and cytochrome P-450.     

膜上斑点洁显示人红细胞b5还原酶活性

人红细胞NADH-细胞色素b₅还原酶是使高铁血红蛋白还原的主要酶类, 其缺陷将导致遗传性高铁血红蛋白血症. 目前, 主要通过分光光度法测定b₅还原酶活性. 我们将b₅还原酶抗体点于硝酸纤维膜上, 以此捕获并富集红细胞胞浆b₅还原酶. 有b₅还原酶活性的斑点用噻唑蓝染色. 此法简单直观, 可用于b₅还原酶的定性和半定量测定, 为遗传性高铁血红蛋白血症的诊断提供了一种新的实验手段.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A method for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography.2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with M_r of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 μmol of cytochrome b, 4 μmol of cytochrome c₁ 7–8 μmol of nonheme iron, corresponding to 3.5–4 μmol of the Rieske iron-sulfur protein, less than 1.0 μmol of ubiquinone and about 60 μmol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2 g of Triton X-100 per g protein.3. Cytochrome b exhibits an α-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. ?10 and +100 mV, at pH 7.0. The absorbance maximum of cytochrome c₁ lies at 553 nm and its half-reduction potential amounts to +250 mV.4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20°C.5. A dissociation constant K′_s of 5.5 mM has been determined for the Triton-solubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas.6. A K′_m value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.     

Participation of a cytochrome b5-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in NADH-semidehydroascorbic acid reductase activity of rat liver

The participation of a cytochrome b₅-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in the NADH-semidehydroascorbate (SDA) reductase activity of rat liver was studied. NADH-SDA reductase activity was strongly inhibited by antibodies against OM cytochrome b and NADH-cytochrome b₅ reductase, whereas no inhibition was caused by anti-cytochrome b₅ antibody. NADH-SDA reductase exhibited the same distribution pattern as OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity among various subcellular fractions and submitochondrial fractions. Both activities were localized in outer mitochondrial membrane. These observations suggest that OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase system participates in the NADH-SDA reductase activity of rat liver.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.     

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b₅ of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 °C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number ( ) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance ( ), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 >cytochrome b₅ >mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114, and ). Thus, the greatest difference in the degree of solubilization of the three cytochromes was observed with Triton X-405 ( and ). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 ( and ).     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Intermembrane electron transport in the absence of added water-soluble carriers

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b₅ reductase or cytochrome b₅ has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems “intact mitochondria — mersalyzed microsomes” and “mersalyzed mitochondria— untreated microsomes”. No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b₅ reductase and/or cytochrome b₅, localized in microsomal and outer mitochondrial membranes.