Enzymic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Biochemistry ◽  
1987 ◽  
Vol 26 (23) ◽  
pp. 7409-7418 ◽  
Author(s):  
N. Elise Gabriel ◽  
Nike V. Agman ◽  
Mary F. Roberts
Keyword(s):  
Phase State ◽  
Matrix Phase ◽  
Short Chain ◽  
Enzymic Hydrolysis ◽  
Unilamellar Vesicles ◽  
Hydrolysis Of ◽  
Long Chain

scholarly journals Partial purification of a diacylglycerol lipase from bovine aorta

1994 ◽  
Vol 298 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M W Lee ◽  
D L Severson
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Short Chain ◽  
Partial Purification ◽  
Diacylglycerol Lipase ◽  
Bivalent Cations ◽  
Triton X ◽  
Hydrolysis Of ◽  
Long Chain

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

scholarly journals Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase

1997 ◽  
Vol 324 (3) ◽  
pp. 941-950 ◽  
Author(s):  
Denis ENGLISH ◽  
Margaret MARTIN ◽  
Kevin A. HARVEY ◽  
Luke P. AKARD ◽  
Ruth ALLEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Molecular Mass ◽  
Biological Systems ◽  
Short Chain ◽  
Intact Cells ◽  
Ability To Act ◽  
Phosphatidic Acids ◽  
Hydrolysis Of ◽  
Long Chain

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 °C and pH 7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1.4×10-3 M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin–Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

scholarly journals Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 320
Author(s):  
Arnaud Masselin ◽  
Antoine Rousseau ◽  
Stéphanie Pradeau ◽  
Laure Fort ◽  
Rodolphe Gueret ◽  
...  
Keyword(s):  
Time Course ◽  
Water Soluble ◽  
Organic Fertilizers ◽  
Symbiotic Associations ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Ecological Transition ◽  
Optimized Conditions ◽  
Hydrolysis Of ◽  
Long Chain

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

Variation in Quantity of Methanol Recovered from Raisins

1963 ◽  
Vol 46 (2) ◽  
pp. 341-343
Author(s):  
M Alice Brown ◽  
James R Woodward ◽  
Floyd DeEds
Keyword(s):  
Methyl Ester ◽  
Esterase Activity ◽  
Enzymic Hydrolysis ◽  
Methanol Content ◽  
Naturally Occurring ◽  
Pectin Esterase ◽  
Hydrolysis Of

Abstract The amount of naturally occurring methanol in fruit must be known so that the quantity left as fumigation residue can be determined. In a study of methanol content of raisins, which had given inconsistent results, the raisins were subjected to different conditions of treatment immediately prior to methanol determination. Conditions that favored pectin esterase activity gave higher values for methanol content than conditions known to inactivate enzymes. Evidence was also obtained that both chemical and enzymic hydrolysis of methyl ester groups of pectic materials occur during analysis.

scholarly journals Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.

1975 ◽  
Vol 64 (3) ◽  
pp. 586-607 ◽  
Author(s):  
N Simionescu ◽  
M Siminoescu ◽  
G E Palade
Keyword(s):  
Plasma Proteins ◽  
Intercellular Junctions ◽  
Rat Diaphragm ◽  
Enzymic Hydrolysis ◽  
Graphic Analysis ◽  
Heme Peptides ◽  
Future Work ◽  
Hydrolysis Of ◽  
Muscle Capillaries

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.

Investigation of the products of an enzymic hydrolysis of collagens

Biochemistry ◽  
1969 ◽  
Vol 8 (12) ◽  
pp. 4716-4723 ◽  
Author(s):  
Howard B. Bensusan
Keyword(s):  
Enzymic Hydrolysis ◽  
Hydrolysis Of

THE HYDROLYSIS OF MONOPHOSPHOINOSITIDE BY EXTRACTS OF BRAIN

1967 ◽  
Vol 45 (6) ◽  
pp. 853-861 ◽  
Author(s):  
W. Thompson
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Calcium Ions ◽  
Brain Extract ◽  
Monovalent Cations ◽  
High Concentration ◽  
Diffusible Factor ◽  
Hydrolysis Of ◽  
The Brain ◽  
Long Chain

The hydrolysis of monophosphoinositide by soluble extracts from rat brain is described. Diglyceride and inositol monophosphate are liberated along with a small amount of free fatty acids. Hydrolysis of the lipid is optimal at pH 5.4 in acetate buffer. The reaction is stimulated by calcium ions or by high concentration of monovalent cations and, to a less extent, by long-chain cationic amphipathic compounds. Enzyme activity is lost on dialysis of the brain extract and can be restored by diffusible factor(s). Some differences in the conditions for hydrolysis of mono- and tri-phosphoinositides are noted.

scholarly journals Constancy of the optimum temperature of an enzyme under varying concentrations of substrate and of enzyme

1914 ◽  
Vol 88 (602) ◽  
pp. 258-262
Keyword(s):  
Experimental Evidence ◽  
Aspergillus Oryzae ◽  
Hydrolytic Enzyme ◽  
Fixed Duration ◽  
Optimum Temperature ◽  
Enzymic Hydrolysis ◽  
Main Interest ◽  
General Law ◽  
The Moment ◽  
Hydrolysis Of

In a recent paper a new enzymic relation is recorded. For the enzymic hydrolysis of salicin—by the enzyme which Gabriel Bertrand and the author have named salicinase —it is found that, in an action of fixed duration, the temperature of greatest activity of the ferment is always the same, whatever the dilutions of substrate and of enzyme adopted for the determination. In other words, the duration of the action being constant, the optimum tem­perature of the ferment is independent of the concentration both of the substrate and of the enzyme. The observation is suggestive: if true of one enzyme it may be true of all, and possibly becomes the enunciation of a general law. Herein, for the moment, lies its main interest. In the present paper further experimental evidence for this hypothesis in given, in the case of another hydrolytic enzyme, the maltase of Aspergillus oryzæ (taka-diastase).

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