scholarly journals Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation

1989 ◽  
Vol 263 (2) ◽  
pp. 565-572 ◽  
Author(s):  
D Riendeau ◽  
D Denis ◽  
L Y Choo ◽  
D J Nathaniel
Keyword(s):  
Lipid Peroxidation ◽  
High Speed ◽  
Chemical Reduction ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Lipoxygenase Activity ◽  
Supernatant Fraction ◽  
Acid Oxidation ◽  
Thiol Compounds ◽  
Stimulation Of

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.

scholarly journals BRAIN MITOCHONDRIA

1963 ◽  
Vol 19 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Diana S. Beattie ◽  
Howard R. Sloan ◽  
R. E. Basford
Keyword(s):  
High Speed ◽  
Brain Cortex ◽  
Lactate Production ◽  
Mitochondrial Fraction ◽  
Brain Mitochondria ◽  
Glycolytic Enzymes ◽  
Supernatant Fraction ◽  
Glycolytic Activity ◽  
High Speed Supernatant ◽  
Stimulation Of

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.

scholarly journals Protein disulphide-isomerase of chick-embryo tendon

1984 ◽  
Vol 219 (1) ◽  
pp. 51-59 ◽  
Author(s):  
B E Brockway ◽  
R B Freedman
Keyword(s):  
Chick Embryo ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
High Speed ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Supernatant Fraction ◽  
Marker Enzyme ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.

Salt tolerance in the halophyte Suaeda maritima : some properties of malate dehydrogenase

1976 ◽  
Vol 273 (927) ◽  
pp. 523-540 ◽  
Keyword(s):  
Sodium Chloride ◽  
Salt Tolerance ◽  
Molecular Mass ◽  
Malate Dehydrogenase ◽  
High Speed ◽  
Gel Filtration ◽  
Supernatant Fraction ◽  
Suaeda Maritima ◽  
Substrate Concentrations ◽  
Mass Form

Malate dehydrogenase activity in a high speed supernatant fraction prepared from homogenates of the shoots of the halophyte Suaeda maritima was separated into two fractions by gel filtration. Sodium chloride stimulated the activity of the larger molecular mass form of the enzyme to a greater extent than that of the smaller molecular mass form : the latter constituted the bulk of the activity. The degree of stimulation by sodium chloride increased with increasing substrate concentrations. Differences between the properties of the enzymes isolated from plants grown with and without sodium chloride are described. The results obtained with the halophyte enzyme were very similar to those obtained with the glycophyte Pisum sativum . Maximum activation of enzyme activity was obtained with a salt concentration of 50 mol m -3 in both species and at higher concentrations activity was inhibited. The results are discussed in relation to the mechanism of salt tolerance in halophytes.

ACTIVATION OF SOLUBILIZED STEROID-TRANSFORMING ENZYMES OF ADRENAL MICROSOMAL ORIGIN BY SERUM PROTEINS

1972 ◽  
Vol 54 (2) ◽  
pp. 297-315 ◽  
Author(s):  
MARGOT A. HAMILTON ◽  
R. W. McCUNE ◽  
SIDNEY ROBERTS
Keyword(s):  
High Speed ◽  
Microsomal Fraction ◽  
Adrenal Glands ◽  
Serum Proteins ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fraction ◽  
Rat Serum ◽  
Microsomal Preparation ◽  
Stimulation Of

SUMMARY The reactions which result in the conversion of pregnenolone to progesterone and of progesterone to deoxycorticosterone in undisrupted microsomal preparations from rat adrenal glands were stimulated by homologous serum. The active materials were shown to be firmly associated with serum proteins. The dialysable fraction of serum was either without effect on these transformations or was inhibitory. The enzyme systems involved were partially solubilized by exposure of the microsomal preparation to prolonged sonic treatment or to 1% Triton N-101. After either treatment, 35–40% of the original specific activity of the steroid 21-hydroxylase system responsible for the conversion of progesterone to deoxycorticosterone was found in the supernatant fraction after high-speed centrifugation. However, this solubilized system did not respond to serum preparations. The same procedures also resulted in a supernatant fluid which showed about 50–60% of the initial specific activity of the multi-enzyme system involved in the conversion of pregnenolone to progesterone. In these instances, the stimulatory effect of serum was retained or accentuated. Acetone powders prepared from the adrenal microsomal fraction were also active in the conversion of pregnenolone to progesterone and responded to serum with enhanced activity. Earlier observations that activation of steroid 21-hydroxylase by homologous rat serum was specific for the β-globulin fraction were confirmed in the present investigations. In contrast, stimulation of the conversion of pregnenolone to progesterone was apparently due principally to the albumin fraction. Albumin preparations from a number of other sources, as well as whole human serum protein, were also effective in this regard. The active protein preparations selectively stimulated 4-ene-3β-hydroxysteroid dehydrogenase activity, but did not activate 5-ene-3-oxosteroid isomerase in the microsomal fraction. This finding suggested that activation of 5-ene-3β-hydroxysteroid dehydrogenase was responsible for stimulation of progesterone synthesis from pregnenolone. The results indicate that the protein-bound factor in rat serum which was capable of stimulating the conversion of progesterone to deoxycorticosterone in microsomal preparations from rat adrenal glands was different from that which activates the conversion of pregnenolone to progesterone. Moreover, these diverse factors appeared to act by different mechanisms.

ENPLATE NI-423

Alloy Digest ◽  
1986 ◽  
Vol 35 (11) ◽  
Keyword(s):  
Wear Resistance ◽  
Physical Properties ◽  
Surface Treatment ◽  
High Speed ◽  
Chemical Reduction ◽  
Electronic Devices ◽  
Electroless Nickel ◽  
Heat Treating ◽  
Corrosion And Wear ◽  
Phosphorus Alloy

Abstract ENPLATE NI-423 is a nickel-phosphorus alloy deposited by chemical reduction without electric current. It is deposited by a stable, relatively high-speed functional electroless nickel process that produces a low-stress coating with good ductility and excellent resistance to corrosion. Its many uses include equipment for chemicals and food, aerospace components, molds and electronic devices. This datasheet provides information on composition, physical properties, and hardness. It also includes information on corrosion and wear resistance as well as heat treating, machining, joining, and surface treatment. Filing Code: Ni-343. Producer or source: Enthone Inc..

Secretagogue-induced changes in subcellular Ca2+ distribution in isolated pancreatic acini

1981 ◽  
Vol 240 (2) ◽  
pp. G130-G140
Author(s):  
R. L. Dormer ◽  
J. A. Williams
Keyword(s):  
Atomic Absorption Spectrometry ◽  
High Speed ◽  
Microsomal Fraction ◽  
Absorption Spectrometry ◽  
Subcellular Fractionation ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Induced Changes ◽  
Stimulation Of

In a prior study, we demonstrated that pancreatic secretagogues increased both the uptake into and washout of 45Ca2+ from isolated mouse pancreatic acini. The net result of these processes was an initial fall in total acinar cell Ca2+ content. In the present study, we have employed subcellular fractionation of acini under conditions that minimized posthomogenization redistribution of Ca2+ in order to localize those organelles involved in intracellular Ca2+ fluxes. Homogenization and differential centrifugation of acini, preloaded with 45Ca2+ and subjected to a period of washout, showed that carbachol induced an increased loss of 45Ca2+ from all fractions isolated. The high-speed microsomal fraction lost 45Ca2+ to a greater extent than did whole acini; measurement of total Ca2+ by atomic absorption spectrometry showed a net loss of Ca2+ from this fraction. Purification of the lower-speed fractions indicated that carbachol increased 45Ca2+ exchange with both zymogen granules and mitochondria, but net Ca2+ levels in these organelles were unchanged. It was concluded that stimulation of pancreatic acini by carbachol results in the release of calcium from a microsomal compartment leading to a rise in cytoplasmic Ca2+, increased exchange with granule and mitochondrial Ca2+, and increased efflux of Ca2+ from the cell.

scholarly journals miR-21-5p regulates mitochondrial respiration and lipid content in H9C2 cells

2019 ◽  
Vol 316 (3) ◽  
pp. H710-H721 ◽  
Author(s):  
Victoria L. Nasci ◽  
Sandra Chuppa ◽  
Lindsey Griswold ◽  
Kathryn A. Goodreau ◽  
Ranjan K. Dash ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Fatty Acid Oxidation ◽  
Lipid Content ◽  
Mitochondrial Respiration ◽  
Acid Oxidation ◽  
H9c2 Cells ◽  
Cellular Lipid ◽  
Transfected Cells

Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

scholarly journals Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver

1989 ◽  
Vol 263 (2) ◽  
pp. 355-363 ◽  
Author(s):  
L Shaw ◽  
R Schauer
Keyword(s):  
Submandibular Gland ◽  
Mouse Liver ◽  
High Speed ◽  
Metal Salts ◽  
Supernatant Fraction ◽  
Inhibitory Influence ◽  
Hydroxylase Activity ◽  
Acetylneuraminic Acid ◽  
Liver Homogenates ◽  
High Speed Supernatant

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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