The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase

Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.     

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.     

Kinetic studies on methemoglobin reduction by human red cell NADH cytochrome b5 reductase.

The intermediate hemoglobins which were produced by the partial reduction of methemoglobin with human red cell NADH cytochrome b5 reductase were fractionated by the preparative isoelectric focusing. These were found to be composed of alpha3+beta2+ and alpha2+beta3+ valency hybrids by the studies of absorption spectra and inositol hexaphosphate-induced difference spectra. Furthermore, the changes in these intermediate hemoglobins during reduction of methemoglobin by the enzyme were studied in the presence or absence of inositol hexaphosphate using the isoelectric focusing fractionation on Ampholine plate gel...     

NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.     

Effects of domain-specific erythrocyte membrane modulators on acetylcholinesterase and NADH:cytochrome b5 reductase activities

Previously, we showed using electron paramagnetic resonance that the physical state of one side of erythrocyte membranes could be modulated by agents which interact with the opposite side (reviewed in Butterfield, 1989, Biological and Synthetic Membranes, A. R. Liss, Inc., New York). The present study was undertaken to determine whether membrane-bound enzymes would exhibit a similar transmembrane modulation effect. The effects of known, domain-specific modulators of the physical state of erythrocyte membranes on the activity of two membrane-bound enzymes were investigated. Acetylcholinesterase, an enzyme having its active site situated on the extracellular side of the membrane, seemed to be unaffected by most of the modulators employed in this study, with the exception of reversible inhibition by benzyl alcohol. Conversely, the activity of NADH:cytochrome b5 reductase, an enzyme whose active site is located on the cytoplasmic side of the erythrocyte membrane, was increased by those agents that interact primarily with skeletal proteins to increase skeletal protein-protein interactions; however, those agents which interact primarily with the skeleton to decrease protein-protein interactions decreased the activity of NADH:cytochrome b5 reductase. This enzyme's activity was also significantly altered by lectins which bind specifically to the external face of glycophorin A on the opposite side of the membrane, but it's activity was unaffected by concanavalin A, a lectin which binds to the external face of band 3. The results of these biochemical studies suggested that NADH:cytochrome b5 reductase can interact with and its activity can be modulated by skeletal or transmembrane proteins. In addition, these results support the hypothesis that in transmembrane signaling processes, biophysical and biochemical changes are correlated.     

The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle

Mitochondria isolated from minute amounts (100-500 mg) of human skeletal muscle displayed a very high rotenone-resistant NADH cytochrome c reductase activity. Moreover, compared to succinate cytochrome c reductase activity, a low rate of rotenone-sensitive NADH cytochrome c reductase activity was measured when using standard procedures to disrupt mitochondrial membranes. Only a drastic osmotic shock in distillated water as a mean to disrupt mitochondrial membrane was found to strongly increase the actual rate of the rotenone-sensitive activity. This was accompanied by a decrease in the rotenone-insensitive activity. Using such a simple procedure, the NADH cytochrome c reductase was found 70-80% inhibited by rotenone and roughly equivalent to 70-85% of the activity of the succinate cytochrome c reductase.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b₅ reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b₅ reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b₅ to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b₅ from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b₅ during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b₅ stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b₅ and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b₅ from rat liver microsomes was shown to inhibit erythrocyte cytochrome b₅ reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b₅ preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b₅ of human erythrocytes are immunochemically similar to NADH-cytochrome b₅ reductase and cytochrome b₅ of liver microsomes, respectively.     

Alterations of mechanical characteristics of human skeletal muscle during strength training

To investigate the influence of strength training on the mechanical characteristics of human skeletal muscle, 14 male subjects went through training of combined heavy concentric and eccentric contractions three times a week for 16 weeks. The strength training program consisted mainly of dynamic exercises for leg extensors with loads of 80 to 120% of one maximum repetition. The force-time curves produced during various vertical jumps were the basis for calculation of various mechanical parameters. In addition to a great increase (p less than 0.001) in maximal isometric force, heavy resistance strength training also caused significant (p less than 0.05-0.01) increases in heights and in various mechanical parameters in positive work phases of vertical and drop jumps. The increase in positive force during a fast dynamic contraction was correlated (p less than 0.01) with the reduced time to produce a certain submaximal force level in isometric condition. No changes in the elastic properties of the muscle were observed as judged from the difference between the counter-movement and squat jumps. When the training was followed by the 8-week detraining period a great decrease (p less than 0.001) in maximal force took place, but only minor changes (ns) were observed in fast force production.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

NADH nitrate reductase and related NADH cytochrome c reductase species in barley

Nitrate and nitrate-less barley (Hordeum vulgare cv Golden Promise) shoot extracts were examined by Sephadex G200 gel filtration and sucrose density gradient analysis and the MWs of NR and CR species present were determined from their Stokes radii and sedimentation coefficients by the method of Siegel and Monty. Nitrate-less plant extracts possessed a CR species of MW 27 800 whilst nitrate-plant extracts possessed CR species of MW 203 000, 61 000, 40 000 and 27 800. The MW 203 000 CR species was associated with NADH-NR, FMNH-NR and MV°-NR activities and represents the NR complex. The MW 40 000 and 61 000 CR species were shown to be derived from the NR complex. We suggest that the MW 40 000 and 61 000 CR species represent either subunits of the NR complex or domains cleaved from the intact NR complex by endogenous proteinases.     

NADH-cytochrome c reductase activity in cultured human lymphocytes: Similarity to the liver microsomal NADH-cytochrome b5 reductase and cytochrome b5 enzyme system

A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b₅ reductase or cytochrome b₅. The activity was linear with cellular homogenates up to 5.2 × 10⁶ cells/ml and had abroad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.     

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.     

The effect of training and detraining on several enzymes in horse skeletal muscle.

Training and detraining had little effect on the activity of glycogen synthase, hexokinase, glycerol 3-phosphate dehydrogenase or total protein. The activity of 3-hydroxyacyl-CoA dehydrogenase increased markedly during training. After 5 weeks of detraining, the activity of 3-hydroxyacyl-CoA dehydrogenase was returning to pre-training values, whilst by 10-week detraining, the levels were increasing again.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.