NADH-cytochrome C-reductase Activity Research Articles

The influence of adrenalectomy on monoamine oxidase and NADH cytochrome c reductase in the rat heart.

The effect of adrenalectomy on the activities of monoamine oxidase (MAO), NADH cytochrome c reductase (NCR), succinate dehydrogenase, malate dehydrogenase, fumarase, NAD+ nucleosidase and acid phosphatase in homogenates of rat hearts was examined. Besides MAO only the NCR activity increased. However, both the total and the rotenone-insensitive NCR activities increased, with that of the rotenone-insensitive being about half of the total, which indicated that the effect of adrenalectomy was exerted on components of this enzyme localized on both the inner and outer membranes of the mitochondrion. The lack of effect on the other enzymes suggests that adrenalectomy has a relatively selective action on MAO and NCR, and does not work by a generalized increase in protein synthesis or by an effect on the FAD cofactor. The MAO increase was seen with a variety of substrates, and was due to a rise in Vmax without change in Km. The response to adrenalectomy in the summer differed from that seen in the winter. The possible reasons for these effects of adrenalectomy are discussed.

Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei)

Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b(5); NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c-reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6+/-6.34 pmol mg(-1) protein (P<0.05 versus control, 136.5+/-7.1 pmol mg(-1) protein) for 2.3 ppt and 305.6+/-8.5 pmol mg(-1) protein (P<0.001 versus control, 132.3+/-6.8 pmol mg(-1) protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150+/-6.5 pmol mg(-1 )protein (P<0.01 versus control, 84.6+/-5.2 pmol mg(-1) protein) for 2.3 ppt and 175+/-5.5 pmol mg(-1) protein (P<0.01 versus control, 87.6+/-5.4 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7+/-0.6 nmol min(-1 )mg(-1) protein (P<0.05 versus control, 6.8+/-0.55 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 12.1+/-0.45 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 6.9+/-0.42 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5+/-0.31 nmol min(-1 )mg(-1) protein (P<0.001 versus control, 4.6+/-0.45 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 9.6+/-0.32 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 4.9+/-0.41 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b(5 )(124.8+/-3.73 pmol mg(-1) protein (P<0.01 versus control, 76.8+/-4.2 pmol mg(-1) protein) for 2.3 ppt and 115.3+/-3.86 pmol mg(-1) protein (P<0.01 versus control, 76.4+/-4.25 pmol mg(-1 )protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110+/-3.11 pmol mg(-1) protein (P<0.01 versus control, 63.7+/-3.24 pmol mg(-1 )protein) for 2.3 ppt and 95.3+/-2.63 pmol mg(-1) protein (P<0.01 versus control, 61.4+/-2.82 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution.

Nitric Oxide Inhibits Electron Transfer and Increases Superoxide Radical Production in Rat Heart Mitochondria and Submitochondrial Particles

Nitric oxide (·NO) released byS-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective ·NO concentration of 0.1 μM, while succinate– and NADH–cytochrome-creductase activities were half-maximally inhibited at 0.3 μM·NO. Submitochondrial particles treated with ·NO (either from GSNO or from a pure solution) showed increased[formula]and H2O2production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with ·NO also showed increased H2O2production. Cytochrome spectra and decreased enzymatic activities in the presence of ·NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone–cytochromebregion of the respiratory chain, the latter leading to the increased[formula]production. Electrochemical detection showed that the buildup of a ·NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of ·NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial[formula]reacts with ·NO to form peroxynitrite and, by removing ·NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of ·NO, inhibition of electron transfer, ·NO-induced[formula]production, and ONOO−formation participate in the regulatory control of mitochondrial oxygen uptake.

Strongyloides ratti: Mitochondrial enzyme activities of the classical electron transport pathway in the infective (L3) larvae

Submitochondrial particles prepared from S. ratti L3 larvae exhibited NADH-oxidase (NOX), NADH-ferricyanide reductase (NFR), NADH-cytochrome- c-reductase (NCR), succinate-cytochrome- c-reductase (SCR), and cytochrome- aa 3 -oxidase activities of 2.1 ± 0.3, 8.9 ± 1.3, 0.6 ± 0.1, 1.0 ± 0.2 and 1.2 ± 0.3 nm min −1 mg protein −1 respectively, at 37°C. The NCR and NOX activities were 39.3% and 23.5% of the NFR activity, suggesting the occurrence of a rate-limiting step or bifurcation of the respiratory electron transport (RET) pathway on the oxygen-side of RET-Complex I. The NCR activity was 50% that of cytochrome- aa 3 -oxidase activity which suggests partitioning of electron flow at the level of RET-Complex III and/or the quinone-function. Antimycin A and rotesone but not 2-thenoyl trifluoreacetone (TTFA) inhibited NCR activity, the EC 50 values were 3.6 × 10 −6 m, 3.7 × 10 −7 m, respectively. SCR activity was inhibited by antimycin A (EC 50 = 3.8 × 10 −6 m) and TTFA (EC 50 = 2.8 × 10 −5 m) but not by rotenone. The results suggest that presence of classical and alternate RET-pathways in S. ratti L3 larvae.

Platelet phosphatidylinositol kinase activity is not altered in Alzheimer disease.

We previously reported a specific decline in phosphatidylinositol (PI) kinase activity in the neocortex of patients with Alzheimer disease (AD) as compared to controls, whereas phosphatidylinositol phosphate (PIP) kinase activity appeared not to be affected (Jolles et al., 1992). In search of a possible systemic effect of AD, in the present study we investigated phosphoinositide kinase activity in platelets from patients with AD and from control subjects. The study was based on the notion that disease-specific abnormalities in the brain could be reflected in blood platelets. PI kinase activity was studied in platelet homogenates and in a salt-solubilized protein fraction of platelets, because of the difference in subcellular localization of the different types of PI kinases. In addition, NADH cytochrome-C reductase was measured in platelet homogenates as a marker for the endoplasmic reticulum, to detect a possible proliferation of the endoplasmic reticulum. AD patients and normal elderly controls showed no difference in PI kinase activity in either enzyme fraction. Furthermore, NADH cytochrome-C reductase activity and the protein/phospholipid ratio per 10(6) platelets were the same for AD patients and controls. This was taken as an indication that platelets in AD patients do not show proliferation of intracellular membranes.

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5′-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-14C-glucose or UDP-14C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl2 was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl2 the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10μM UDP-glucose in the presence of 3 mM MnCl2 was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 μM UDP-xylose in the presence of 3 mM MnCl2, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-14C-glucose, and fractions enriched in Golgi apparatus, both (1→3)- and (1→4)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (1→3)-glucosyl. With the substrate, UDP-14C-xylose, mostly (1→4)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.

Aflatoxin - induced alteration in the levels of membrane chemical of subcellular organelles isolated from excised, incubated glycine max, cv. "Essex" roots. I. Non-enriched organelles.

Isolates of aflatoxin-producing strains of Aspergillus grow on autoclaved and field-grown (lesser extent) Glycine max beans. Both mixed and aflatoxin B1 inhibit G. max, cv. ‘Essex’ bean germination and elongation of either attached or excised cultured roots. Because B1 impairs the latter roots' ability to intracellularize [14C]-leucine, it may alter plasmalemma structure and/or function. To determine whether incubation of excised roots for 18 hours in toxin-containing medium could affect cellular membrane chemical content, organelles were isolated by differential centrifugation (1 000, 40 000, and 80 000 xg) of homogenates and characterized chemically. Statistically significant differences between treated and untreated roots in acid insoluble protein but not either sterol or lipid phosphorus levels were observed for both 40,000 and 80,000 xg pellets. Protein and sterol recoveries were 81 (treated) and 84 (untreated) % for the former and 77 (treated) and 79 (untreated) % for the latter. Lipid phosphorus recoveries were 87.3 (treated) and 136 (untreated) % with and 96 (treated) and 83 (untreated) without membrane stabilization. Protein:sterol:lipid phosphorus were 35.7∶4.5∶1 (1 000 xg), 18.9∶3.6∶1 (40000 xg), 26.3∶4.6∶1 (80 000 xg) and 1,010∶29∶1 (80 000 xg supernatant) for untreated and 36.9∶3.3∶1 (1,000 xg), 23.1∶3.8∶1 (40 000 xg), 36.2∶4.8∶1 (80 000 xg) and 1,053∶21.7∶1 (80 000 xg supernatant) for treated roots. Significant differences in RNA content between treated and untreated roots were found for both 1 000 and 40 000 xg pellets but not for the 80 000 xg pellet and its supernatant. Whereas a significant increase in the 1 000 xg pellet occurred upon treatment, a decrease was noted for the 40 000 xg pellet but not for the 80 000 xg pellet and its supernatant. Similar pH 6 (plasmalemma marker enzyme) and 9 (mitochondrial marker enzyme) K+-stimulated ATPase activities were demonstrated for 40 000 and 80 000 xg pellets. The 1 000 xg pellet contained greater than 50% of the NADH-cytochrome c-reductase activity (endoplasmic reticulum marker enzyme) recovered from fractions examined for this activity which was absent from the 40 000 xg pellet. Both the 80 000 xg pellet and its supernatant possessed equivalent reductase activities. Inosine diphosphatase activity (dictyosome marker enzyme) was not present in 1 000 xg pellets obtained from either treated or untreated roots but was in both 40 000 and 80 000 xg pellets. Based on these results, a tentative assignment of organelles to each fraction (xg force) is reported.

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R(F) value of 0.12 to an R(F) of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R(F) value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R(F) value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.

RESPIRATORY ENZYMES IN THE HEART AND LIVER OF THE PRENATAL AND POSTNATAL RAT.

1. Heart and liver tissue samples were obtained from rats in various developmental stages from the 12-day-old embryo to the 120-day-old postnatal animal. 2. The body, heart and liver weights and percentage protein in the liver and heart of the prenatal and postnatal rat were determined. 3. The activities of NADH-, NADPH- and succinate-cytochrome c reductases and cytochrome oxidase were determined also. 4. The specific activities of all the enzymes increased in both heart and liver during late foetal development (16 days to term). The NADH- and succinate-cytochrome c-reductase activities in the heart increased threefold during the neonatal period (0 to 25 days post partum) and then remained constant to 120 days. All reductase activities increased in the liver three- to six-fold during the neonatal period. Cytochrome-oxidase activity in both tissues increased sixfold during this time but plateaued in the liver at 12 days rather than 25 days. 5. A sex difference was observed in NADH-cytochrome c-reductase activity in the liver. Up to 25 days post partum the activity was the same in both sexes, but from that time on the activity continued to increase in the female but remained unchanged in the male. 6. NADPH-cytochrome c-reductase activity increased only in the liver. 7. These results indicate that different electron-transport pathways predominate according to the tissue, developmental stage and sex of the animal.