Presence Of Pyridine Nucleotide Research Articles

Increase in NAD(P)H‐dependent generation of active oxygen species and changes in lipid composition of microsomes isolated from roots of zinc‐deficient bean plants

The role of zinc (Zn) in maintaining the structural and functional integrity of plant membranes was investigated in the present work. The relationship between the activity of NAD(P)H oxidases generating active oxygen species and changes in lipid composition and peroxidation was evaluated in microsomal membrane vesicles isolated from roots of Zn‐defícient bean (Phaseolus vulgaris L., cv. Bobis) plants. Zinc content of bean root microsomal membranes was decreased by about 30% by Zn deficiency. Microsomes isolated from roots of Zn‐deficient plants showed higher rates of NAD(P)H oxidation and NAD(P)H‐dependent O2 ‐ generation than Zn‐sufficient roots. Microsomal O2 ‐ consumption, measured in the presence of pyridine nucleotides, was also considerably enhanced by Zn deficiency. This latter activity was greatly stimulated by Fe(III)EDTA, while inhibited by Superoxide dismutase (SOD) and catalase, indicating that active oxygen species were produced during the oxygen consuming enzyme reaction. Zinc deficiency caused a decline in microsomal phospholipid (PL) content. In addition, saturated fatty acids were present at a higher proportion than unsaturated fatty acids in microsomes from Zn‐deficient roots. Sterol content of microsomal vesicles was also modified by Zn deficiency, which led to an increase in the planar sterol campesterol and a concomitant decrease in stigmasterol and sitosterol content. NADPH‐dependent lipid peroxidation, directly measured in microsomal vesicles as malondialdehyde (MDA) production, was slightly enhanced by Zn deficiency. These results support the idea that Zn deficiency determines an enhanced generation of harmful oxygen species by membrane‐associated enzymes and show that this activity can be more pronounced in the presence of iron (Fe), which accumulates in Zn‐deficient tissues. The relationship between the occurrence of this phenomenon and the changes in membrane lipid profile is discussed.

Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells

High-affinity 3,3',5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).

Existence of acetyl-CoA-dependent chain elongation system in hepatic peroxisomes of rat: Effects of clofibrate and di-(2-ethylhexyl)phthalate on the activity

The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5–7.0 and that for the mitochondrial activity was pH 7.5–8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 m m N-ethylmaleimide or 1 m m p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 m m KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD + into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the β-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the β-oxidation system under physiological conditions.

Evidence for the nonenzymatic and irreversible binding of cytembena to rat liver microsomes in vitro

Characteristics of the irreversible binding of cytembena (CYT) to rat liver microsomal proteins have been investigated in vitro. Binding of [ 14C]CYT to rat liver microsomal proteins remained unchanged in the presence of SKF-525A or after heat denaturation and was not dependent upon the presence of pyridine nucleotide (NADPH or NADH). Ethacrynic acid, cysteine, dithiothreitol, and lysine were found to block the irreversible binding of [ 14C]CYT to microsomal proteins in a dose-dependent manner. The rank order of effectiveness as inhibitors of CYT binding was ethacrynic acid > cysteine > dithiothreitol > lysine. In other studies, CYT was shown to preferentially form adducts with cysteine rather than lysine or glycine. Using structural analogs and metabolites of CYT, it was found that 4-methoxybenzoylacrylic acid and β-benzoylacrylic acid were effective competitors of [ 14C]CYT binding to liver microsomal proteins. By contrast, 4-methoxybenzoylpropionic acid, 5-(4-methoxyphenyl)dihydro-2(3 H)furanone and 4-hydroxy-4-(4-methoxyphenyl)butyric acid were ineffective as inhibitors of the irreversible binding of CYT. These data suggest that sulfhydryl groups are involved in the nonenzymatic binding of CYT and that the presence of a carbon-carbon double bond in CYT, in debrominated metabolites or structural analogs, is requisite for an interference of the binding of CYT to microsomal proteins.

Glutamate formed from lignoceric acid by rat brain preparation in the presence of pyridine nucleotide and cytosolic factors: a brain-specific oxidation of very long chain fatty acids.

Glutamate formed from lignoceric acid by rat brain preparation in the presence of pyridine nucleotide and cytosolic factors: a brain-specific oxidation of very long chain fatty acids.

Biosynthesis of monoterpenes: Conversion of the acyclic precursors geranyl pyrophosphate and neryl pyrophosphate to the rearranged monoterpenes fenchol and fenchone by a soluble enzyme preparation from fennel ( Foeniculum vulgare)

(+)-(1 S)-Fenchone, an irregular bicyclic monoterpene ketone thought to be derived via rearrangement of a bicyclic precursor, is one of the major terpenoids of the volatile oil of fennel ( Foeniculum vulgare M.). A soluble enzyme preparation obtained from homogenates of young fennel leaves converted the acyclic precursors geranyl pyrophosphate and neryl pyrophosphate to fenchol in the presence of Mn 2+, and in the presence of pyridine nucleotide, dehydrogenated the fenchol formed to fenchone. Particulate enzyme fractions were inactive in the synthesis of fenchane-type monoterpenes. The identities of the biosynthetic products as fenchol and fenchone were confirmed by radiochromatographic analysis and by the synthesis of crystalline derivatives. Differential crystallization of the fenchyl hydrogen phthalates and the fenchyl p-nitrobenzoates demonstrated that the initial cyclic product was endo-[ 3H]fenchol and not the exo-epimer, while resolution of [ 3H]fenchone oxime via the diastereomeric l-menthyloxymethyl ethers confirmed the stereochemistry of the labeled cyclic products as (−)-(1 S)- endo-fenchol and (+)-(1 S)-fenchone. Biosynthetic l-endo-[ 3H]fenchol derived from the [1- 3H]-labeled acyclic precursors was chemically converted to (+)-(1 R)-α-fenchocamphorone, and exchange of the α-protons of this derivative established that 3H was located specifically on the bridge carbon (C-7) of the original fenchane nucleus. Thus, the bicyclic fenchane skeleton was shown to be derived by rearrangement of a pinane-type intermediate. These results provide strong evidence that fenchone is derived by the cyclization of geranyl pyrophosphate or neryl pyrophosphate to endo-fenchol followed by dehydrogenation of this bicyclic alcohol, and they constitute the first demonstration of the biosynthesis of a rearranged monoterpene in a cell-free system.

Inability of the testosterone-estrogen-binding globulin to catalyze the oxidation or reduction of steroids in the presence of pyridine nucleotides.

The testosterone–estrogen-binding globulin of human serum was examined for 17β-hydroxy-steroid dehydrogenase and 5α-reductase activities. None was found.

NAD +-induced phosphate acceptor specificity in submitochondrial systems

The nonspecific phosphorylation reaction in submitochondrial systems obtained by sonication became much more specific in the presence of pyridine nucleotides. Phosphorylation of IDP was inhibited by increasing concentrations of NAD +, an inhibition which became biphasic upon the addition of hexokinase (EC 2.7.1.1), whereas phosphorylation of ADP was totally unaffected. The inhibition by NAD + was of a noncompetitive type with K i = 1.5 · 10 −4 M. The nicotinamide part of the molecule was immediately responsible for the inhibitory effect. Although rotenone or antimycin A interfered with the specificity promoted by NAD +, the pyridine nucleotide effect was not due to the introduction of an energy-competing NAD + reduction. No evidence was found of an initiated selective nucleoside triphosphatase reaction, so the regulation was considered to take place at a primary event of the energy conservation mechanism and possibly exerted by an allosteric change. It is suggested that endogenous pyridine nucleotides which are lost upon sonic disintegration of mitochondria may constitute a factor for regulation of the nucleoside specificity during oxidative phosphorylation.

The Kinetic Properties of Spinach Leaf Glyoxylic Acid Reductase

Abstract The kinetic properties of spinach leaf glyoxylic acid reductase have been evaluated. In the presence of pyridine nucleotides, the enzyme catalyzes the reversible reduction of glyoxylate to glycolate and hydroxypyruvate to d(-)-glycerate. The pH optima of the reductive reactions are between 6.0 and 6.5; the pH optima of the oxidative reactions are at 8.9. The enzyme is competitively inhibited by pyruvate, 3-mercaptopyruvate, and 3-fluoropyruvate and noncompetitively inhibited by dihydroxyfumarate. A spectral shift in protein absorbance, from 280 mµ to 270 mµ, is associated with the dihydroxyfumarate inhibition. Ternary rate equations of the enzyme and a degraded form of the enzyme have been calculated and compared. Specific changes in the Michaelis constant for pyridine nucleotides suggest that the degraded form has undergone structural modifications in the pyridine nucleotide binding site. All forms of the enzyme are anion-regulated in the direction of d(-)-glycerate or glycolate formation. Phosphate stimulates hydroxypyruvate reduction at all anion concentrations; chloride, bromide, sulfate, and nitrate stimulate at low concentrations but inhibit at high. The anion effects are pH-dependent and are competitive with hydroxypyruvate or glyoxylate. They are noncompetitive with reduced pyridine nucleotides. Evidence is presented which suggests that a ligand-induced conformational change is important in the functioning of anion effectors.

Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease.

The leukocytes of patients with chronic granulomatous disease (CGD) may be identified by their failure to reduce Nitro Blue Tetrazolium (NBT) during phagocytosis. This reaction, normally detected in the phagocytic vacuole, is absent or delayed in CGD monocytes and eosinophils as well as in neutrophils, even though sonicates of normal and CGD leukocytes contain equal activities of a cyanide insensitive enzyme system capable of reduction of NBT in the presence of pyridine nucleotide. Enlargement of CGD phagocytic vacuoles appears to be inhibited. Histochemical estimates of the rate of release of alkaline phosphatase are normal in CGD cells. Peroxidase activity is released from CGD cells, but the rate appears to be somewhat slower than normal in some cases. The latter observation may be explained by the increased intensity of the peroxidase stain in resting and phagocytizing CGD cells. The severity of the defect in NBT reduction within the phagocytic vacuoles of the leukocytes of patients and carriers is more variable than was previously appreciated. Some female carriers have profoundly reduced dye reduction and others are nearly indistinguishable from normal. Three brothers with CGD demonstrated significant, albeit delayed, NBT reduction in phagocytic vacuoles during prolonged incubation of their leukocytes. No obvious relationship exists, however, between the rate of reduction of NBT in vacuoles and the clinical severity of the disease.

Studies on the cyclophorase system. XVIII. The oxido-reductions of glycolysis

Summary The oxidative enzymes of glycolysis are present in cyclophorase preparations. Their activity is not increased by the presence of pyridine nucleotides. When a small quantity of supernatant is added to the gel, presumably to supply the nonoxidative enzymes, it can oxidize glucose and various intermediates of the glycolysis scheme to carbon dioxide and water. D (-)-Lactic acid is oxidized by cyclophorase preparations in the absence of supernatant, but supernatant is required for the oxidation of L (+)-lactic acid.