Cytochalasin E-induced oxidative metabolism in polymorphonuclear leukocytes.

The extent of the cyanide-resistent oxidative burst of polymorpho-nuclear leukocytes after stimulation with cytochalasin E was shown to depend markedly on the osmolarity of the cell-suspension medium. With granulocyte concentrations up to 2 X 10(6) cells/ml, optimal oxygen consumption and releases of H2O2 and superoxide anions were reached at 180 mOsmol and 2 X 10(-5) M cytochalasin E. After removal of unbound activator, the cellular oxidative activity remained unaltered and continued to depend on the used osmotic conditions. It is proposed that binding of cytochalasin to the plasma membrane induces an irreversible activation of the oxidative system, whereas the resulting metabolic activity depends on conformational changes in the plasma membrane.     

Vascular oxidative metabolism under different metabolic conditions.

Control of respiration in vascular smooth muscle was examined while the metabolic state of the tissue was manipulated. During KCl-induced contractures in the presence of 5 mM glucose, oxygen consumption increased by 10 nmol/per min g without any decrease in phosphocreatine (PCr) or ATP as determined by 31P-NMR indicating a control of respiration which does not involve changes in high-energy phosphates (e.g., ADP, phosphorylation potential). However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2-deoxyglucose as the sole substrate, oxygen consumption increased 7.4 nmol/min per g while PCr decreased by more than 50% (resulting in a 2-fold increase in the calculated free ADP) with no change in tension from resting tone. During a subsequent KCl induced contracture in the presence of 2-deoxyglucose, oxygen consumption increased an additional 7.2 nmol/min per g while PCr continued to decline. Therefore, at least two mechanisms of respiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phosphates.     

Heme metabolism and oxidative stress

The role of heme metabolism in oxidative stress development and defense reactions formation in mammals under different stress factors are discussed in the article. Heme metabolism is considered as the totality of synthesis, degradation, transport and exchange processes of exogenous heme and heme liberated from erythrocyte hemoglobin under erythrocyte aging and hemolysis. The literature data presented display normal heme metabolism including mammals heme-binding proteins and intracellular free heme pool and heme metabolism alterations under oxidative stress development. The main attention is focused to the prooxidant action of heme, the interaction of heme transport and lipid exchange, and to the heme metabolism key enzymes (delta-aminolevulinate synthase and heme oxygenase), serum heme-binding protein hemopexin and intracellular heme-binding proteins participating in metabolism adaptation under the action of factors, which cause oxidative stress.     

Hypoxia and mitochondrial oxidative metabolism

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria's central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O₂] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F₁F₀ ATPase in the hypoxia-induced mitochondrial autophagy.     

L-Tyrosine oxidative metabolism byStreptomyces aureofaciens

Summary L-Tyrosine metabolism found in normal mammalian system via homogentisic acid was investigated inStreptomyces aureofaciens S-834 as L-tyrosine was shown to possess the stimulatory role in the production of chlortetracycline antibiotic. L-Tyrosine as well as the substrates of the intermediary oxidative enzymes-p-hydroxyphenylpyruvate (p-HPPA) hydroxylase and homogentisate (HGA) oxygenase were incubated with the washed, resting cells of the culture and by adding sodium diethyldithiocarbamate and,-dipyridyl, the specific inhibitors of these enzymes to the culture, the oxidative intermediates-p-HPPA and HGA were blocked from further utilisation and were accumulated and isolated by extraction with the appropriate solvent system. The intermediates consisted of keto acids and aromatic phenolic acids were separated by adsorption and elution on silica gel and sephadex gel columns. p-HPPA and acetoacetate (AA) were identified as keto acids by obtaining their 2,4-dinitrophenylhydrazone derivatives. HGA as well as p-HPPA were identified as phenolic acids along with HGA lactone using diazotized reagents. These investigations helped to establish L-tyrosine oxidative pathway inStreptomyces aureofaciens via p-HPPA, HGA and AA leading to the biosynthesis of chlortetracycline antibiotic.     

Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.     

Methotrexate: studies on cellular metabolism. II--Effects on mitochondrial oxidative metabolism and ion transport

Effects of methotrexate (MTX) on mitochondrial oxidative metabolism and ion transport were studied. MTX decreases the membrane potential (delta psi) upon energization of the mitochondrial membrane by NAD+-linked substrates and decreases the amplitude and velocity of swelling induced by glutamate and alpha-ketoglutarate. MTX also has an inhibitory effect on the activities of the oxidation enzymes of NAD+-linked substrates without interfering with the oxidation systems of FAD-linked substrates. The effects of MTX could be interpreted as a consequence of a decrease in the ionic conductivity of the mitochondrial inner membrane.     

Lead inhibits oxidative metabolism of macrophages exposed to macrophage-activating factor.

1. An improved rat spleen perfusion is described incorporating a method of defibrination which avoids the use of heparin and enables the spleen to be perfused with rat blood for several hours at a haematocrit of 40% and for 12 h or more at a haematocrit of 20%. 2. Glucose oxidation accounted for 11.6% of the total oxygen consumption but this represented only 8% of total glucose uptake, which was largely converted to lactate and released into the perfusate. However, significant amounts of lactate were oxidized. These results can be explained by the presence of at least two cell populations, one emphasizing the anaerobic oxidation of glucose and the other aerobic metabolism, particularly of lactate. 3. Non-esterified fatty acid and 3-hydroxybutyrate, when available at physiological concentrations, were shown to be major oxidative fuels of the spleen. 4. Chylomicron triacylglycerol was hydrolysed readily and taken up, but not oxidized extensively.     

The selection of Chinese hamster cells deficient in oxidative energy metabolism.

A selection scheme based on the nutritional requirements of a previously described respiration-deficient Chinese hamster line has been used to isolate new mutants defective in oxidative energy metabolism. Three of the primary characteristics of this type of mutant are (1) a strict dependency on the continued presence of glucose for survival; (2) a drastic reduction in the rate of oxygen consumption; (3) an inhibition of Krebs cycle activity resulting in auxotrophy for asparagine and carbon dioxide. In the case of one cell line which was used (V79), up to 65% of the survivors of a selection were found to possess this phenotype after only one round of selection. By contrast, it proved much more difficult to obtain such mutants from another cell line (CCL16). A preliminary characterization of a number of these mutants is presented.     

Potassium, neuroglia, and oxidative metabolism in central gray matter.

Reviewed is the author's investigation of potassium in extracellular fluid of cerebral neocortex and spinal cord determined with ion-selective microelectrodes, and of oxidative metabolism monitored by fluorometric determination of intramitochondrial NADH in intact cortex. When gray matter is excited by afferent input, or by direct electrical stimulation, the logarithm of the rise of extracellular potassium concentration ([K+]0), the sustained shift of electrical potential, and the response of oxidative metabolism are linearly correlated. However, during seizures and during spreading depression, the correlation is broken, suggesting that the demand for oxidative energy exceeds that corresponding to the elevation of [K+]0. There exists a critical concentration of [K+]0 at which spreading depression inevitably erupts (12 mM for cat cerveau isole), but no such critical level could be detected for seizures. The rate of clearance of excess potassium from extracellular fluid is slower for high concentrations than for low; this rate is further slowed by the administration of phenobarbital, and possibly also of diphenylhydantoin. Changes of membrane potential of glia cells in the mammalian spinal cord can adequately be described by the Nernst equation.     

Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.