Inhibition Of Aconitase Activity Research Articles

Differential mitochondrial dinitrosyliron complex formation by nitrite and nitric oxide

Nitrite represents an endocrine reserve of bioavailable nitric oxide (NO) that mediates a number of physiological responses including conferral of cytoprotection after ischemia/reperfusion (I/R). It has long been known that nitrite can react with non-heme iron to form dinitrosyliron complexes (DNIC). However, it remains unclear how quickly nitrite-dependent DNIC form in vivo, whether formation kinetics differ from that of NO-dependent DNIC, and whether DNIC play a role in the cytoprotective effects of nitrite. Here we demonstrate that chronic but not acute nitrite supplementation increases DNIC concentration in the liver and kidney of mice. Although DNIC have been purported to have antioxidant properties, we show that the accumulation of DNIC in vivo is not associated with nitrite-dependent cytoprotection after hepatic I/R. Further, our data in an isolated mitochondrial model of anoxia/reoxygenation show that while NO and nitrite demonstrate similar S-nitrosothiol formation kinetics, DNIC formation is significantly greater with NO and associated with mitochondrial dysfunction as well as inhibition of aconitase activity. These data are the first to directly compare mitochondrial DNIC formation by NO and nitrite. This study suggests that nitrite-dependent DNIC formation is a physiological consequence of dietary nitrite. The data presented herein implicate mitochondrial DNIC formation as a potential mechanism underlying the differential cytoprotective effects of nitrite and NO after I/R, and suggest that DNIC formation is potentially responsible for the cytotoxic effects observed at high NO concentrations.

Effects of sodium metavanadate on in vitro neuroblastoma and red blood cells

Toxicity of vanadium on cells is one of the less studied effects. This prompted us to study the structural effects induced on neuroblastoma and erythrocytes by vanadium (V) sodium metavanadate. This salt was incubated with mice cholinergic neuroblastoma cells and intact human erythrocytes. To learn whether metavanadate interacts with membrane lipid bilayers it was incubated with bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). These are phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Exposure of neuroblastoma cells to metavanadate showed significant decreases in cell viability as well as in cell number correlating with inhibition of aconitase activity. In scanning electron microscopy (SEM) and defocusing microscopy (DM) it was observed that induced on erythrocytes the formation of echinocytes. However, no effects were obtained when metavanadate was made to interact with DMPC and DMPE multibilayers and liposomes, assays performed by X-ray diffraction and differential scanning calorimetry (DSC), respectively. These results imply that the effects of metavanadate on erythrocytes are through interactions with proteins located in the membrane outer moiety, and could still involve other minor lipid components as well. Also, partly unsaturated lipids could interact differently the fully saturated chains in the model systems.

Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme's activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.

Succinic acid monomethyl ester protects rat pancreatic islet secretory potential against interleukin-1β (IL-1β) without affecting glutamate decarboxylase expression or nitric oxide production

Rat pancreatic islets exposed to interleukin-1β (IL-1β) in the presence of succinic acid monomethyl ester (SAM) have a higher insulin release in response to glucose and higher glucose oxidation rates, as compared to islets exposed to IL-1β alone. These beneficial effects of SAM were not accompanied by any decrease in IL-1β-induced nitric oxide (NO) production nor inhibition of aconitase activity. Moreover, SAM did not increase biosynthesis of glutamate decarboxylase. SAM apparently improves β-cell function mostly by increasing the capacity of these cells to endure NO exposure and partial blockage of the Krebs cycle.

Cytokines induce an L-arginine-dependent effector system in nonmacrophage cells.

Treatment of EMT-6 mammary adenocarcinoma cells with gamma interferon (rMuIFN gamma) plus tumor necrosis factor (rMuTNF alpha) and/or interleukin-1 (rHuIL-1 alpha) causes release of iron-55 label, inhibition of DNA replication, and inhibition of aconitase activity. In addition, the same combinations of cytokines induce EMT-6 cells to synthesize L-citrulline, nitrite, and nitrate directly from L-arginine. Lipopolysaccharide (LPS) can act as a cofactor in the induction of these metabolic effects when added to EMT-6 cells in the presence of rMuIFN gamma. The results show that increased levels of cytokines in the microenvironment can induce a novel effector pathway in somatic cells not specialized for host defense, resulting in specific metabolic effects as well as the inhibition of cellular proliferation.

Aconitase and citric acid fermentation by Aspergillus niger.

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.

Inhibition by aconitine of aconitase of pig heart

1. The present paper describes the in vitro inhibition type, inhibition constant and inhibition rate of pig heart aconitase by aconitine. 2. The inhibition of aconitase activity by aconitine is totally non-competitive. The datum of inhibition constant, K i = 0.11 ± 0.01 l/ l, indicates high affinity of aconitine by aconitase. 3. The rate constant of inhibition k 1 = 49 min −1, the half-life for aconitase inhibition of l min and the 50% inhibition time of 8 min by 0.1 mmol/l aconitine show high specific inhibition of the enzyme by the inhibitor. 4. These results suggest a possible molecular reason for the toxic and pharmacological actions produced on experimental animals by aconitine.

A study of the kinetics of aconitase inhibition and activation

1. 1. The inhibition of aconitase activity by chelating agents has been studied over a wide range of substrate concentrations. The chelating agents, o-phenanthroline, pyrophosphate, 8-hydroxyquinoline, and α,α′-dipyridyl inhibit aconitase activity, whereas EDTA has a slight activating effect. Excess substrate will not reverse the inhibition. 2. 2. Activation experiments indicate that ferrous ions and substrate may combine independently with the enzyme. 3. 3. Aconitase can utilize a ferrous-aconitate chelate as substrate and shows greater activity in the presence of this chelate than in the presence of substrate alone. A ferrous-isocitrate chelate can also be utilized but the enzyme has an equal or higher affinity for free isocitrate than the isocitrate-iron chelate.