Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

1988 ◽  
Vol 66 (5) ◽  
pp. 425-435 ◽  
Author(s):  
Amy Mok ◽  
Tanya Wong ◽  
Octavio Filgueiras ◽  
Paul G. Casola ◽  
Don W. Nicholson ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Mitochondrial Activity ◽  
Rat Lung ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Mercuric Ions

CDPdiacylglycerol pyrophosphatase (E. C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6–8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 μM and Vmax values of 311 and 197 pmol∙min−1∙mg protein−1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

Sinapine Esterase I. Characterization of Sinapine Esterase from Cotyledons of Raphanus sativus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 715-720 ◽  
Author(s):  
Gerhild Nurmann ◽  
Dieter Strack
Keyword(s):  
Enzyme Activity ◽  
Esterase Activity ◽  
Raphanus Sativus ◽  
Sinapic Acid ◽  
Inhibitory Effect ◽  
Ph Optimum ◽  
Diisopropyl Fluorophosphate ◽  
E 600 ◽  
Red Radish

Abstract From cotyledons of Raphanus sativus (red radish) an esterase activity which catalyzes the hy­drolysis of sinapine into sinapic acid and choline has been isolated. The enzyme, which has a near absolute specificity, is not analogous with any esterase described in the literature. The reaction has a pH optimum of 8.5 and the apparent Km is 1.95 × 10-5 m. The enzyme is relatively insensi­tive to both physostigmine (eserine) {Ki = 1.73 × 10-4 m) and neostigmine (Ki = 2 .1 3 × 10-4 ᴍ). Diisopropyl fluorophosphate (DFP) showed no inhibition and diethyl p-nitrophenylphosphate (E 600) only a slight inhibitory effect at 10-5 ᴍ, respectively. Choline (10-2 ᴍ) was inhibitory but acetylcholine (10-2 ᴍ) stimulated the enzyme activity.

Characterization of Hexokinase and Fructokinase from Suspension-Cultured Catharanthus roseus Cells

1988 ◽  
Vol 43 (11-12) ◽  
pp. 827-834 ◽  
Author(s):  
Yüko Yamashita ◽  
Hiroshi Ashihara
Keyword(s):  
Catharanthus Roseus ◽  
Divalent Cations ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Phosphate Donor

Abstract Two different hexose-phosphorylating enzymes, hexokinase and fructokinase, were partially purified from suspension-cultured Catharanthus roseus cells. One of the enzymes, hexokinase, catalyzed the phosphorylation of both glucose and fructose. The Km values for glucose and fructose were 0.06 mM and 0.23 mM, respectively. The Vmax of the enzyme with fructose was approximately three times higher than with glucose. This enzyme was specific in its requirement for ATP and its Km value for ATP was 52 μM. The optimum pH was 8.0 and Mg2+ or Mn2+ was required for the activity. The activity was inhibited by considerably higher concentrations of ADP (i.e., 4 mM ADP was required for 50% inhibition). The second enzyme, fructokinase, was specific for fructose, and no activity was detected with glucose as substrate. This enzyme used UTP or CTP as phosphate donor. The Km values of this enzyme for fructose and UTP were 0.13 mM and 0.15 mM, respectively. The pH optimum was 7.2, and Mg2+ or Mn2+ was required for the activity. These divalent cations could be partially replaced by Ca2+. The activity was inhibited noncompetitively by ADP and AMP. 90% inhibition of the activity by 0.5 mM ADP was observed in the presence of 2 mM UTP and 5 mM MgCl2. Fructose-2,6-bisphosphate, glucose-1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate had little or no effect on the activity of both the hexokinase and the fructokinase. Based on these results, a discussion is presented of the role of hexokinase and fructokinase and their involvement in the regulation of the metabolism of sugars in Catharanthus cells.

scholarly journals External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of α and γ Subunits and Reducing Channel Open Probability

2002 ◽  
Vol 277 (51) ◽  
pp. 50098-50111 ◽  
Author(s):  
Shaohu Sheng ◽  
Clint J. Perry ◽  
Thomas R. Kleyman
Keyword(s):  
Divalent Cations ◽  
Wild Type Mouse ◽  
Inhibitory Effect ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Open Probability ◽  
Histidine Residues ◽  
Low Concentrations ◽  
High Concentrations ◽  
A Site

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni2+on cloned mouse α-β-γ ENaC expressed inXenopusoocytes. Ni2+reduced amiloride-sensitive Na+currents of the wild type mouse ENaC in a dose-dependent manner. The Ni2+block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni2+was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni2+inhibition of the remaining current. Mutations at αHis282and γHis239located within the extracellular loops significantly decreased Ni2+inhibition of ENaC currents. The mutation αH282D or double mutations αH282R/γH239R eliminated Ni2+block. All mutations at γHis239eliminated Ni2+-induced inward current rectification. Ni2+block was significantly enhanced by introduction of a histidine at αArg280. Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni2+block. Although αH282C-β-γ channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), α-β-γ H239C channels were insensitive to MTSET. From patch clamp studies, Ni2+did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni2+reduces ENaC open probability by binding to a site consisting of αHis282and γHis239and that these histidine residues may participate in ENaC gating.

Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization

2004 ◽  
Vol 82 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Emi Mishiro ◽  
Ming-Yih Liu ◽  
Yoichi Sakakibara ◽  
Masahito Suiko ◽  
Ming-Cheh Liu
Keyword(s):  
Molecular Cloning ◽  
Divalent Cations ◽  
Functional Characterization ◽  
The Other ◽  
Inhibitory Effects ◽  
Amino Acid Sequence Level ◽  
Ph Optimum ◽  
Rapid Amplification ◽  
High Degree

By employing the reverse transcriptase – polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.Key words: zebra fish, tyrosylprotein sulfotransferase, molecular cloning.

Purification and Characterization of Three Extracellular Proteinases Produced by Pseudomonas fluorescens INIA 745, an Isolate from Ewe's Milk

1999 ◽  
Vol 62 (5) ◽  
pp. 543-546 ◽  
Author(s):  
J. FERNÁNDEZ ◽  
A. F. MOHEDANO ◽  
P. GAYA ◽  
M. MEDINA ◽  
M. NUÑEZ
Keyword(s):  
Pseudomonas Fluorescens ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Extracellular Proteinases ◽  
Ewe's Milk

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45°C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded β-casein, with preference to αs1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.

scholarly journals Peroxidative damage of mitochondrial respiration is substrate-dependent

2009 ◽  
pp. 685-692
Author(s):  
R Endlicher ◽  
P Křiváková ◽  
H Rauchová ◽  
H Nůsková ◽  
Z Červinková ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Peroxidative Damage ◽  
Palmitoyl Carnitine ◽  
Low Concentrations ◽  
Tert Butyl Hydroperoxide ◽  
Strong Inhibitory Effect

The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 µM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 µM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage.

Separation, Partial Purification and Characterization of a Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato Fruits

1982 ◽  
Vol 37 (3-4) ◽  
pp. 165-173 ◽  
Author(s):  
P. Schreier ◽  
G. Lorenz
Keyword(s):  
Fatty Acid ◽  
Inhibitory Effect ◽  
Fatty Acid Hydroperoxide ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isoelectric Points ◽  
Acid Hydroperoxide ◽  
Membrane Bound

Abstract A membrane-bound enzyme catalysing the cleavage of 13-hydroperoxy-(Z)-9,(E)-11-oc-tadecadienoic acid (13-LHPO) and 13-hydroperoxy-(Z)-9,(E)-11,(Z)-15-octadecadienoic acid (13-LnHPO) to C6-aldehydes was isolated and partially purified from apples and tomatoes. Attempts to employ Ultrogel AcA 34 and AcA 22 in a gel chromatographic purification step were partially frustrated by reaggregation phenomena. However, by using Sepharose CL-4 B an enzyme fraction (MW 200 000 Da) with lipoxygenase and fatty acid hydroperoxide cleaving activity could be separated from a high molecular-weight active eluate. By applying preparative isoelec­ tric focussing to the tomato protein we succeeded in separating the fatty acid cleaving activity from the lipoxygenase, because o f their different isoelectric points of pH 5.8 -6 .1 and pH 5.0, respectively, An 8.4-fold purification of the fatty acid cleaving activity was achieved. A pH-optimum of 5.5 and a Km-value of 2.6 × 10-5 м/1 for the 13-hydroperoxide of linoleic acid were measured. p-Chloromercuribenzoic acid (1 mм) showed significant inhibitory effect on the fatty acid hydroperoxide cleaving enzyme, but no evidence o f inhibition was found with 1 mм H2O2, KCN, DABCO and EDTA or superoxide dismutase (270 U). The maximum amount of fatty acid hydroperoxide decomposition (C6-aldehyde formation) was determined to be 59%.

Purification and Partial Characterization of Deoxyribonuclease I from Bovine Parotid Gland

1977 ◽  
Vol 56 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Roger L. Lundblad ◽  
Steve Hoffman ◽  
Claudia M. Noyes ◽  
Henry S. Kingdon
Keyword(s):  
Parotid Gland ◽  
Proteolytic Enzymes ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Acid Extraction ◽  
Ph Optimum ◽  
Deoxyribonuclease I ◽  
Dnase A

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNase's.

scholarly journals STUDIES OF LUNG METABOLISM

1966 ◽  
Vol 30 (1) ◽  
pp. 45-57 ◽  
Author(s):  
Oscar K. Reiss
Keyword(s):  
Electron Microscope ◽  
Adenosine Triphosphatase ◽  
Protein Concentration ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Antimycin A ◽  
Magnesium Ions ◽  
Rat Lung ◽  
Morphological Criteria

The isolation and partial characterization of subcellular particles from rabbit and rat lung are described. Detailed methods for separating a purified, active mitochondrial fraction are outlined and evaluated in terms of enzymatic, chemical, and morphological criteria. Mitochondrial preparations from rabbit and rat liver were used as comparative indices. The lung mitochondrial fraction was identified by its ability to oxidize succinate with a P/O ratio of 1.7 by a process sensitive to 2,4 dinitrophenol and antimycin A. The adenosine triphosphatase activity of the lung mitochondrial fraction is stimulated by magnesium ions, but this stimulation is not augmented by 2,4 dinitrophenol. In the absence of magnesium ions, the specific activity of the adenosine triphosphatase increases with increasing protein concentration. The presence of lysosomes in the mitochondrial fraction is suggested by acid phosphatase and cathepsin activities and by electron microscope observations.

Isolation and characterization of a mitochondrial D-amino acid oxidase from Neurospora crassa

1977 ◽  
Vol 55 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Melvin G. Rosenfeld ◽  
Edward H. Leiter
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Mitochondrial Fraction ◽  
Acid Oxidation ◽  
Gel Chromatography ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Ph Optimum ◽  
Isolation And Characterization

D-Amino acid oxidase (EC 1.4.3.3) activity in homogenates of Neurospora crassa strain SY7A was found to sediment with the mitochondrial fraction. Digitonin fractionation studies on purified mitochondria have indicated a matrix localization of the enzyme. Additionally, a peroxidase (EC 1.11.1.7) activity, which may remove hydrogen peroxide formed as a product of D-amino acid oxidation, was also found in the mitochondrial matrix.Partial purification (20- to 30-fold) of the mitochondrial D-amino acid oxidase was achieved. The enzyme exhibited a pH optimum between 9.0 and 9.2, temperature optimum between 20 and 30 °C, and a molecular weight of 118 000 ± 6000 as determined by gel electrophoresis and 125 000 as determined by gel chromatography.

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