scholarly journals Phosphatidylinositol phosphodiesterase in higher plants

1980 ◽  
Vol 192 (1) ◽  
pp. 279-283 ◽  
Author(s):  
R F Irvine ◽  
A J Letcher ◽  
R M C Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Higher Plants ◽  
Water Soluble ◽  
Apium Graveolens ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Detectable Activity ◽  
Ph Range

1. The lower regions of the stem of celery (Apium graveolens L.) contain a soluble enzyme that hydrolyses phosphatidylinositol. 2. The lipoidal product of hydrolysis is diacylglycerol, and the water-soluble products are 1:2-cyclic phosphoinositol and phosphoinositol in the approximate proportions of 60% and 40% respectively: this indicates that a phosphodiesterase (phospholipase C-like) activity is cleaving the phosphatidylinositol. 3. The enzyme requires a bivalent cation, Ca2+ being the most effective activator. 4. The enzyme has a pH optimum, depending on conditions of assay, of pH 5.9-6.6 and in this pH range shows no detectable activity against phosphatidylcholine or phosphatidylethanolamine. 5. The activity is stimulated by phosphatidic acid and slightly inhibited (30% at concentrations equimolar with phosphatidylinositol) by phosphatidylcholine. 6. The phosphodiesterase was also detected (but not quantified) in the tips of the flowers in cauliflowers, in outer leaves of onion and in the elongating stem of daffodils. 7. The enzyme's properties are compared with equivalent mammalian enzymes, and its possible role in the catabolism of phosphatidylinositol in higher plants is discussed.

Formation of radioactive diacylglycerol from radioisotope-labelled phosphatidylethanolamine by Escherichia coli extracts

1989 ◽  
Vol 67 (6) ◽  
pp. 288-292 ◽  
Author(s):  
H. Aubry ◽  
P. Proulx
Keyword(s):  
Escherichia Coli ◽  
Phospholipase C ◽  
Substrate Concentration ◽  
Incubation Medium ◽  
Isotope Ratio ◽  
Enzyme Concentration ◽  
Water Soluble ◽  
Ph Optimum ◽  
Direct Incorporation

Radioisotope-labelled phosphatidylethanolamine can be converted to radioactive diacylglycerol in the presence of added unlabelled diacylglycerol. With [14C-glycerol; 3H-acyl]phosphatidylethanolamine as substrate, the conversion to double-labelled diacylglycerol occurred without change in isotope ratio indicating that the whole diacylglycerol moiety of phosphatidylethanolamine was directly involved. With [3H-acyl; 32P]phosphatidylethanolamine, formation of [3H]diacylglycerol occurred without production of labelled water-soluble products and consequently no phospholipase C activity could be detected. Under similar conditions, a conversion of [14C-acyl]- or [3H-acyl]-diacylglycerol to labelled phosphatidylethanolamine could also be shown even in the presence of hydroxylamine. [14C-Glycerol; 3H-acyl] diacylglycerol was converted to double-labelled product without change in isotope ratio which again indicated a direct incorporation of the entire diacylglycerol molecule into phosphatidylethanolamine. Both types of conversions were dependent upon time, enzyme concentration, and substrate concentration, and both displayed a pH optimum of approximately 6 and required no added cofactors. In the presence of increasing concentrations of [14C-acyl]diacylglycerol, added to incubation medium containing [3H-acyl]phosphatidylethanolamine, equal amounts of [14C]phosphatidylethanolamine and [3H]diacylglycerol were formed which matched the decrease in [3H]phosphatidylethanolamine. From these results, we conclude that Escherichia coli has an enzyme that catalyses the exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol, with complete sparing of the phosphoethanolamine moiety.Key words: diacylglycerol, phosphatidylethanolamine, exchange, Escherichia coli.

scholarly journals Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate

1992 ◽  
Vol 286 (3) ◽  
pp. 693-699 ◽  
Author(s):  
M C Chabot ◽  
L C McPhail ◽  
R L Wykle ◽  
D A Kennerly ◽  
C E McCall
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Water Soluble ◽  
Incubation Mixture ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Agonist Stimulation

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

scholarly journals Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase

1971 ◽  
Vol 125 (4) ◽  
pp. 991-997 ◽  
Author(s):  
R. D. Howland ◽  
A. Burkhalter ◽  
A. J. Trevor ◽  
S. Hegeman ◽  
D. Y. Shirachi
Keyword(s):  
Rat Liver ◽  
Uridine Diphosphate ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Ionic Detergent ◽  
Michaelis Constants

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.

scholarly journals Preparation of immobilized baker's-yeast glucose 6-phosphate dehydrogenase attached to modified sepharose and sephadex and a comparison of the properties of these preparations with those of the soluble enzyme

1976 ◽  
Vol 157 (2) ◽  
pp. 289-294 ◽  
Author(s):  
M A Goheer ◽  
B J Gould ◽  
D V Parke
Keyword(s):  
Immobilized Enzyme ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
Enzyme Preparations ◽  
Ph Range ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.

The oxidation of manganous ions under acid conditions by an acidophilous actinomycete from acid soil

Soil Research ◽  
1978 ◽  
Vol 16 (1) ◽  
pp. 91 ◽  
Author(s):  
SM Bromfield
Keyword(s):  
Manganese Oxide ◽  
Acid Soil ◽  
Acid Soils ◽  
Water Soluble ◽  
Organic Media ◽  
Manganese Oxidation ◽  
Ph Optimum ◽  
Acid Media ◽  
Acid Ph ◽  
Ph Range

A Stveptomyces sp. capable of oxidizing manganous ions in soil agar to manganese oxide over the pH range 5-6.5 was isolated from an acid (pH 5.0) soil. The organism failed to grow on organic media above pH 6.5 and had a pH optimum for growth of 5-5.5. On acid media its growth was restricted by manganous ions, and these were deposited as the oxide some distance from the colonies. It is suggested that the function of manganese oxidation is to protect the organism from inhibiting levels of manganous ions. The organism produced, under acid conditions (pH 5.0) in the presence or absence of added manganous ions, a water-soluble extracellular substance that could rapidly oxidize manganous ions to manganese oxide. These observations are discussed in relation to the manganese status of acid soils.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Structural analysis of oligomeric fructans from excised leaves of Lolium temulentum

2020 ◽  
Author(s):  
Ian Sims ◽  
CJ Pollock ◽  
R Horgan
Keyword(s):  
Mass Spectrometry ◽  
Structural Analysis ◽  
Higher Plants ◽  
Water Soluble ◽  
Lolium Temulentum ◽  
Neutral Water ◽  
Alditol Acetates ◽  
Partially Methylated

Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants. © 1992.

scholarly journals A shift in the optimum pH of phospholipase D produced by activating long-chain anions

1969 ◽  
Vol 112 (5) ◽  
pp. 795-799 ◽  
Author(s):  
R. H. Quarles ◽  
R. M. C. Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Activity Profile ◽  
Ph Optimum ◽  
Surface Ph ◽  
Electrophoretic Mobilities ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Long Chain

1. The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) towards ultrasonically treated phosphatidylcholine or large phosphatidylcholine particles activated with ether was maximal near pH5, and there was little activity above pH6. 2. When the enzyme was activated by the addition of phosphatidic acid to large phosphatidylcholine particles the pH optimum was shifted to pH6·5 irrespective of the amount of activator added. 3. When the enzyme was activated with low concentrations of dodecyl sulphate the pH optimum was 5·5 with little activity above pH6. With higher concentrations of dodecyl sulphate the pH–activity profile was shifted upwards towards a pH optimum of 6·5–6·6, the magnitude of the shift depending on the extent of the hydrolysis. 4. The shifts in the pH–activity profiles cannot be correlated with changes in the ‘surface pH’ of the substrate particles calculated from the measurement of their ζ-potentials (electrophoretic mobilities).

Pyridine–Adenine Dinucleotide Transhydrogenase Activity in Cells Cultured from Rat Hepatoma

1972 ◽  
Vol 50 (5) ◽  
pp. 447-456 ◽  
Author(s):  
C. De Luca ◽  
R. P. Gioeli
Keyword(s):  
Phosphate Buffer ◽  
Pyridine Nucleotide ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Apparent Lack ◽  
Minimal Deviation ◽  
Ph Range ◽  
Pyridine Nucleotide Transhydrogenase ◽  
Rat Hepatoma ◽  
Cells Cultured

Preparations from cells cultured from a minimal-deviation hepatoma in the rat exhibit pyridine nucleotide transhydrogenase (NAD(P)H: NAD(P) oxidoreductase, EC 1.6.1.1) activity. The pH optimum, its release by digitonin, and its apparent lack of dependence on steroids for activity tentatively classify it as a transhydrogenase of the type first described for animal tissue.Enzyme preparations from digitonin-treated homogenates were very unstable. The time necessary for the loss of one-half the activity was 16–18 h when the enzyme was stored at 5 °C; this was reduced to 4 h when storage was in polycarbonate tubes.The enzyme apparently transferred hydrogen directly and with equal ease from NADH to both the 3-acetyl-pyridine and thionicotinamide analogues of NAD. Half-saturation values for NAD and its acetylpyridine analogue were 0.99 × 10−5 M and 3.55 × 10−4 M, respectively. The enzyme exhibited its maximum activity in phosphate buffer at pH 5.8. It was inhibited by 50–60% over the pH range 7.0–8.5 in Tris buffer. This could be reversed by dithiothreitol; reversal was complete between pH 8.0 and 8.5.

scholarly journals Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines

1981 ◽  
Vol 197 (2) ◽  
pp. 523-526 ◽  
Author(s):  
Paul D. Wightman ◽  
Mary Ellen Dahlgren ◽  
James C. Hall ◽  
Philip Davies ◽  
Robert J. Bonney
Keyword(s):  
Phospholipase C ◽  
High Activity ◽  
Peritoneal Macrophages ◽  
Ph Optimum ◽  
Reaction Velocity ◽  
Neutral Ph ◽  
Maximum Reaction ◽  
Mouse Peritoneal Macrophages ◽  
Identification And Characterization

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

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