Some kinetic properties of Mucor rouxii phosphofructokinase. Effect of cyclic adenosine 3', 5'-monophosphate.

Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O₂^? is an important intermediate in H₂O₂ formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O₂^t-, but might also help to regulate the level of the important antimicrobial agent, H₂O₂.     

Vanadate-dependent oxidation of pyridine nucleotides in rat liver microsomal membranes

An enzymatic Na₃VO₄-dependent system for the oxidation of reduced pyridine nucleotides in purified rat liver microsomes was characterized. The system has a pH optimum of 6.5, and appears to be specific for vanadate, since activity in the presence of a related transition metal, molybdate, was not detected. Vanadate-dependent oxidation occurred with a concomitant consumption of O₂ and, contrary to previous reports, preferred NADPH over NADH. At pH 6.5, the NADPH/NADH oxidase activity ratio was greater than 2:1. Sodium vanadate-dependent oxidation of NADH was inhibited by rotenone, antimycin A, NaN₃, and NaCN. Conversely, Na₃VO₄-dependent NADPH oxidation was slightly affected by rotenone, but was insensitive to antimycin A, NaN₃, NaCN, or quinacrine. Vanadate-dependent oxidation of either pyridine nucleotide was inhibited by the addition of either Superoxide dismutase or catalase, indicating that both superoxide and hydrogen peroxide may be intermediates in the process. Linear sucrose gradient purification of the microsomes showed that the vanadate-dependent system for NADPH oxidation resides primarily in the endoplasmic reticulum. These studies indicate the existence of separate and distinct enzymatic systems for vanadate-stimulated oxidation of NADPH and NADH in mammalian microsomal membranes, and argue against an exclusive role of endogenous Superoxide in the process.     

Production of Insulin-like Growth Factors and Their Binding Proteins in Primary Cultures of Rat Liver Parenchymal and Nonparenchymal Cells

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells.

Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191–1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-l into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu₂cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-l. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu₂cAMP, and triiodothyronine (T₃), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-l was increased by Bu₂cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations.

These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Evidence for an alternative and non-phosphorylating pathway for NADH reoxidation in a yeast strain resistant to glucose repression

A yeast strain (SP1) resistant to glucose repression modified simultaneously in the fermentative and in the oxidative pathways (loss of alcohol dehydrogenase I and over production of cytochrome a + a3, being insensitive to the glucose effect) developed a secondary mitochondrial hydrogen pathway. Oxidative phosphorylation was measured with exogenous NADH as substrate on mitochondria derived from repressed or derepressed cells. In this strain, antimycin A promotes a partial inhibition of NADH oxidation but a complete inhibition of phosphorylation. Amytal partially inhibits oxidation of NADH but not phosphorylation. KCN inhibits NADH oxidation in a biphasic way (first level 0.1 mM, second level 5 mM) but phosphorylation was fully inhibited by 0.1 mM KCN. This alternative but non-phosphorylating pathway is insensitive to salicyl hydroxamate. The external NADH dehydrogenase, like cytochrome c oxidase is partially insensitive to catabolite repression. These results provide evidence for the presence in strain SP1 of an alternative mitochondrial pathway, going from the external NADH dehydrogenase to an oxidase, different from the normal NADH dehydrogenase ubiquinone pathway.     

Duroquinone-stimulated NADH oxidase and b type cytochromes in the plasma membrane of cauliflower and mung beans

Microsomal membrane preparations of cauliflower inflorescences and mung bean hypocotyls possess duroquinone (DQ)-stimulated NADH oxidase activities at rates of 1–10 nmol NADH · min⁻ · mg⁻. These redox reaction are associated with the endoplasmic reticulum (ER) and the plasma membrane (PM) as shown by the distributions of marker enzymes in sucrose gradients. The NADH oxidase thus partially cosediments with a specific blue light (or ascorbate) reducible b type cytochrome of the PM.Cauliflower membranes are further purified by means of an aqueous polymer two phase method. The NADH oxidase in this presumptive PM fraction is to some extent stimulated by Triton X-100 and insensitive to KCN (1 mM) or quinacrine (0.4 mM). Kinetics for DQ stimulation showed a biphasic saturation curve. These membranes also have a high FeCN reduction capacity induced by NADH but insensitive to DQ.No evidence could be found in the present study for the involvement of the specific b type cytochrome in the NADH dehydrogenase system.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

The inhibition of exogenous NAD(P)H oxidation in plant mitochondria by chelators and mersalyl as a function of pH

The oxidation of exogenous NADH by Jerusalem artichoke ( Helianthus tuberosus L.) tuber mitochondria was strongly inhibited at pH 7.2 by EDTA, EGTA and mersalyl and by chlorotetracycline in the presence of Ca²⁺. This inhibition disappeared at pH 5.5 where about 50% activity was found as compared to controls at pH 7.2. The rate of oxidation of NADPH at pH 5.5 was the same as for NADH but it was inhibited by 50% by both EDTA and mersalyl.
Mitochondria from Arum maculatum spadices oxidised NADH and NADPH with pH optima of 7.2 and 6.5, respectively. In the presence of EDTA the optima shifted to 6.7 and 5.9, respectively, due to an inhibition at higher pH and a lack of inhibition at lower pH. At pH 6.7 NADH oxidation was completely insensitive to both EDTA and mersalyl whereas the oxidation of NADPH was inhibited by more than 50%. The inhibition of NAD(P)H oxidation by chelators at neutral pH was due to the removal of Ca²⁺ from the membranes in both types of mitochondria. The differences observed in the properties of NADH and NADPH oxidation suggest that two different dehydrogenases are involved. Because of the strong pH-dependence and the changes in chelator-sensitivity in the physiological pH-range 6–8 it is suggested that the properties of NAD(P)H oxidation provide the cell with important means of metabolic regulation.     

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1 mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 μM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q₁ and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q₁ and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.     

Reduced pyridine nucleotide-dependent N-hydroxy amine oxidase and reductase activities of hepatic microsomes

The metabolism of a number of primary and secondary hydroxylamines by hepatic microsomes is described. A cyanide-insensitive, reduced pyridine nucleotide-dependent hydroxylamine reductase activity that is independent of oxygen concentration catalyzes the reduction of hydroxylamine and a number of its mono- and disubstituted derivatives to the parent amines. At the pH optimum of 6.3 for the reductase, NADH is the preferred cofactor. The enzyme does not catalyze the reduction of 4-hydroxyaminoquinoline-1-oxide (HAQO) or of 1- or 2-naphthylhydroxylamine, the only known carcinogenic hydroxylamines tested. A hydroxylamine oxidase activity that requires both oxygen and reduced pyridine nucleotide oxidizes only disubstituted hydroxylamines, and the apparent initial product is the corresponding nitrone. Most nitrones undergo immediate hydrolysis in aqueous solution. At the pH optimum of 7.6 for the oxidase, NADPH is the preferred cofactor. NADPH cannot be replaced by a hydrogen peroxide-generating system, and the reaction is not affected by the addition of large amounts of exogenous catalase. Of the various organs which were assayed, the liver contained the greatest amount of both the reductase and oxidase activities; and the major portion of both activities in liver homogenates was found in the microsomal fraction. The two activities respond differently to agents such as deoxycholate, n-octylamine, and sulfhydryl inhibitors, indicating that the reduction and oxidation of the hydroxylamines are catalyzed by different enzymes or enzyme systems. Both activities are insensitive to carbon monoxide and N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation.     

Macrophage metabolism: activation of NADPH oxidation by phagocytosis

Rabbit and guinea pig peritoneal and alveolar macrophages and rabbit polymorphonuclear leucocytes (PMN) have been tested for their capacity to oxidize NADPH and NADH. In all these cells granule-bound NADPH oxidase is much more active than NADH oxidase, thus confirming our previous observations on human blood and guinea pig PMN. If the phagocytes are challenged with bacteria, the activity of NADPH oxidase is considerably stimulated. The enhancement of the oxidase activity is due to an increase of its V_max and, in the case of the PMN, also to a decrease of the K_m. We conclude that NADPH oxidase might play a relevant role in the metabolic stimulation of both PMN and macrophages by phagocytosis.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.     

Inhibition of Cyanide-Insensitive Respiration in <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> SYSU-011 by 8-Hydroxyquinolone

The inhibition of the cyanide (KCN)-insensitive respiration of Klebsiella oxytoca SYSU-011 by 8-hydroxyquinoline (8-HQ) was determined. Results showed that the profile of the rate of oxygen uptake of normal-grown and 8-HQ–grown K. oxytoca SYSU-011 was biphasic and similar, suggesting that 8-HQ did not inhibit the respiration of normal-grown K. oxytoca SYSU-011. A different biphasic KCN inhibition profile of respiration was observed for KCN-grown cells treated with and without 8-HQ. No decrease in respiration rate of KCN-grown cells and a 40% decrease in respiration rate of KCN-grown cells treated with 8-HQ were observed when KCN concentration was 10^–1 mM. Comparing differences of the profiles of oxygen uptake in KCN-grown cells with and without 8-HQ addition indicated that 8-HQ inhibited expression of the KCN-insensitive pathway carried out by nonheme oxidase. Greater inhibition of NADH oxidase activity by 2-n-heptyl-4-hydroxyquinoline-N-oxide from the cell membrane of the KCN-grown cells treated with 8-HQ, and more H₂O₂ production from these cells with than without 8-HQ, suggest that the function of the cyanide-insensitive pathway can stabilize the respiration of the cyanide-grown cells to prevent the production of H₂O₂.     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Effect of sulfite on the energy metabolism of mammalian tissues in correlation to sulfite oxidase activity

Mammalian tissues show significant differences in the activity of sulfite oxidase (EC 1.8.3.1) which detoxifies sulfite by oxidation to sulfate. Lung tissue and phagocytic cells such as alveolar macrophages, peritoneal macrophages, Kupffer cells and granulocytes show very low activities of sulfite oxidase. Liver tissue and hepatocytes, however, exhibit high activities of sulfite oxidase. Lung tissue and macrophages show an almost 100% decrease of the intracellular ATP levels when incubated with 1 mM sulfite at pH 6 for 30 min. In addition, the O₂ consumption of lung tissue is inhibited by 1 mM sulfite at pH 6 by more than 80%. This sulfite-induced decrease of the ATP level and of the O₂ consumption of lung tissue is enhanced between pH 6.0 and pH 7.4 with decreasing pH value of the incubation medium. In contrast, the ATP levels in liver tissue and hepatocytes are not affected by 1 mM sulfite at pH 6. The O₂ consumption of liver tissue and hepatocytes is significantly increased by sulfite due to the high activities of sulfite oxidase. Therefore, the activity of the ‘sulfite-detoxifying enzyme’ sulfite oxidase and the sensitivity of the energy metabolism to sulfite show a reciprocal relationship in the tissues and cells studied.     

Schistosoma mansoni and Biomphalaria glabrata: Ultrastructural localization of enzymes with diaminobenzidine in larvae and host digestive glands.

All mitochondria contained reaction product when daughter sporocysts of Schistosoma mansoni and digestive glands of the snail host, Biomphalaria glabrata, were cytochemically incubated for 45 or 60 min with alkaline 3, 3′-diaminobenzidine (DAB) at pH 7.4 and 9.0. The pigment marked the presence of cytochrome c-cytochrome oxidase activity, and was not found in parasite or gland tissues incubated with DAB and KCN at pH 7.4, 9.0, and 9.8.After incubation for 45 min in the pH 7.4 DAB medium, tegumental mitochondria in young intrasporocyst cercariae showed DAB reaction product, but little or none of the pigment was found in tegumental mitochondria of older, glycocalyx-covered cercariae. In contrast, mitochondria of subtegumental cells were strongly DAB positive at all stages of intrasporocyst cercarial development. No differences in DAB reactivity were detected in mitochondria of sporocysts, or of infected and uninfected host gland cells.Reaction product was found in certain vacuoles of digestive cells incubated in the pH 9.8 DAB medium with KCN, but not in the pH 9.8 DAB medium with amino triazole, or in the pH 7.4 DAB medium. No peroxisomes or microperoxisomes were found in the tissues studied.