NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

A nonfunctional protein in human leukemia cells reacts with antiserum to dihydrofolate reductase from L1210 leukemia cells

Antiserum raised in chickens to dihydrofolate reductase purified from L1210 leukemia cells by affinity chromatography inhibited the catalytic activity and the binding of methotrexate by the enzyme. Lysates of human chronic myelogenous leukemia cells, which had neither catalytic activity for dihydrofolate reductase nor binding of methotrexate, blocked the inhibiting effect of the antiserum on the function of the enzyme in L1210 cell lysates. In double immunodiffusion, these human leukemia cell lysates formed a single precipitin line against the antiserum. These findings indicate that nonfunctional dihydrofolate reductase in human leukemia cells share an antigenic determinant(s) with a functional form of the enzyme from L1210 murine leukemia cells.     

Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.     

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.     

Reconstitution of the monooxygenase system in a solution and in an immobilized phospholipid layer

To clarify the molecular organization of NADH- and NADPH-dependent microsomal redox systems their isolated purified carriers were incorporated into immobilized azolectin layer with a higher viscosity than that of the liposomes. It was shown that the NADH-cytochrome c reductase activity characterizing the NADH-cytochrome b5 reductase and cytochrome b5 interaction sharply decreased in the immobilized system as compared to that in solution. However, the activity of hydroxylase reactions catalyzed by immobilized NADPH-cytochrome P-450 reductase and cytochrome P-450 was the same as in solution. This, the reconstitution in the immobilized phospholipid layer allowed to characterize NADH-cytochrome b5 reductase as a system operating on occasional collisions of its components. On the contrary, the diffusion of the NADPH-dependent redox chain carriers was not the rate-limiting step of the reaction.     

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. IV. Purification and characterization of NADH-cytochrome b5 reductase.

The presence of NADH-cytochrome b5 reductase [EC 1.6.2.2] in microsomes from anaerobically grown yeast was confirmed by its isolation and purification. The purified preparation of the reductase showed an apparent molecular weight of 27,000 daltons. The reductase appeared to contain loosely-bound FAD as a prosthetic group. The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. The reductase, however, could not reduce cytochrome c. Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 micron M, respectively, and optimal pH for cytochrome b5 reduction was 5.6. Although some differences exist between the properties of NADH-cytochrome b5 reductase from yeast and from mammalia, the results indicate a functional similarity of the present enzyme to mammalian NADH-cytochrome b5 reductase in the microsomal electron-transport system.     

A new C-nucleoside analogue of tiazofurin: synthesis and biological evaluation of 2-beta-D-ribofuranosylimidazole-4-carboxamide (imidazofurin)

2-Beta-D-ribofuranosylimidazole-4-carboxamide, an imidazole analogue of the antitumor agent tiazofurin, was synthesized and evaluated for the growth inhibitory activity of human myelogenous leukemia K562 cells.     

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.     

Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli

Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin. Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme. The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme. The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme. Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.     

Ion transport and respiratory control in vesicles formed from reduced nicotinamide adenine dinucleotide coenzyme Q reductase and phospholipids.

NADH-coenzyme Q reductase from bovine heart mitochondria (complex I) was incorporated into phospholipid vesicles by the cholate dialysis procedure. Mixtures of purified phosphatidylcholine and phosphatidylethanolamine were required. Oxidation of NADH by coenzyme Q1 catalyzed by the reconstituted vesicles was coupled to proton translocation, directed inward, with an H+/2e ratio greater than 1.4. Similar experiments measuring proton translocation in submitochondrial particles gave an H+/2e ratio of 1.8. The proton translocation in both systems was not seen in the presence of uncoupling agents and was in addition to the net proton uptake from the reduction of coenzyme Q1 by NADH. Electron transfer in the reconstituted vesicles also caused the uptake of the permeant anion tetraphenylboron. The rate of electron transfer by the reconstituted vesicles was stimulated about 3-fold by uncouplers or by valinomycin plus nigericin and K+ ions. The results indicate that energy coupling can be observed with isolated NADH-coenzyme Q reductase if the enzyme complex is properly incorporated into a phospholipid vesicle.     

Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q10 Reductase in the Plasma Membrane

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ₁₀ and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ₁₀ reductase activity and its internal sequence being identical to cytochrome b ₅ reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ₁₀ content in isolated plasma membranes. We show here the role of CoQ₁₀ and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b ₅-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ₁₀ and its NADH-dependent reductase.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

NADH-cytochrome c reductase, succinate cytochrome c reductase and phospholipids.

Phospholipid peroxidation of isolated rat liver inner mitochondrial membranes induced by either ascorbate or cysteine was accompanied by a release of flavins and coenzyme Q. A straight correlation between this release and the alteration of molecular species of phosphatidylcholine and phosphatidylethanolamine containing one saturated and one unsaturated fatty acid has been found. Peroxidation induced on molecular species of phosphatidylcholine and phosphatidylethanolamine containing only unsaturated fatty acids were accompanied by losses in enzyme activities of NADH-cytochrome c reductase and succinate cytochrome c reductase.     

Cytoplasmically inherited respiratory deficiency of a mouse fibroblast line which is resistant to rutamycin.

Mouse fibroblasts resistant to the drug rutamycin were isolated and found also to be respiratory deficient. These cells produce large amounts of lactic acid, and oxygen consumption data indicate that the first complex of the electron transport chain, NADH-coenzyme Q reductase, is defective. Levels of rotenone-sensitive NADH-cytochrome c reductase and pyruvate decarboxylase of the pyruvate dehydrogenase complex are markedly depressed in the mutant cells. Other components of the electron transport chain appear to be fully functional. The mutant cells were enucleated and fused with another cell line, and the resulting cybrid demonstrated a similar pattern of respiratory deficiency as did the original mutant. These results indicate that this defect in respiration is a cytoplasmically inherited characteristic in this cell line.     

Structural and functional relationships of enzyme activities induced by nitrate in barley

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH(2)-nitrate reductase and NADH-cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH-nitrate reductase (8S), one band of FMNH(2)-nitrate reductase activity (8S) and three bands of NADH-cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH-cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH-cytochrome c reductase band co-sediments with both NADH-nitrate reductase activity and FMNH(2)-nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH-cytochrome c reductase. 4. FMNH(2)-nitrate reductase activity is more stable (t((1/2)) 12.5min) than either NADH-nitrate reductase activity (t((1/2)) 0.5min) or total NADH-cytochrome c reductase activity (t((1/2)) 1.5min) at 45 degrees C. 5. NADH-cytochrome c reductase and NADH-nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH(2)-nitrate reductase activity. 6. Tungstate prevents the formation of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities, but it causes superinduction of NADH-cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH-cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH-nitrate reductase, FMNH(2)-nitrate reductase and NADH-cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH-cytochrome c reductase activity.     

The importance of plasma membrane coenzyme Q in aging and stress responses

The plasma membrane of eukaryotic cells is the limit to interact with the environment. This position implies receiving stress signals that affects its components such as phospholipids. Inserted inside these components is coenzyme Q that is a redox compound acting as antioxidant. Coenzyme Q is reduced by diverse dehydrogenase enzymes mainly NADH-cytochrome b₅ reductase and NAD(P)H:quinone reductase 1. Reduced coenzyme Q can prevent lipid peroxidation chain reaction by itself or by reducing other antioxidants such as α-tocopherol and ascorbate. The group formed by antioxidants and the enzymes able to reduce coenzyme Q constitutes a plasma membrane redox system that is regulated by conditions that induce oxidative stress. Growth factor removal, ethidium bromide-induced ρ° cells, and vitamin E deficiency are some of the conditions where both coenzyme Q and its reductases are increased in the plasma membrane. This antioxidant system in the plasma membrane has been observed to participate in the healthy aging induced by calorie restriction. Furthermore, coenzyme Q regulates the release of ceramide from sphingomyelin, which is concentrated in the plasma membrane. This results from the non-competitive inhibition of the neutral sphingomyelinase by coenzyme Q particularly by its reduced form. Coenzyme Q in the plasma membrane is then the center of a complex antioxidant system preventing the accumulation of oxidative damage and regulating the externally initiated ceramide signaling pathway.     

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.