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The Journal of Biological Chemistry Nov 1980Deoxycholate treatment of horse platelets previously labeled in their phospholipids with [14C]arachidonate produces selective conversion of [14C]phosphatidylinositol...
Deoxycholate treatment of horse platelets previously labeled in their phospholipids with [14C]arachidonate produces selective conversion of [14C]phosphatidylinositol (PI) to [14C]1,2-diacylglycerol. This phospholipase C activity, which has a pH optimum of 7.5, is specific for phosphatidylinositol since other phospholipids or neutral lipids are not affected. Although exogenous Ca2+ is not required for activity, ethylene glycol bis(beta-aminoethyl ether)N,N,N',N-tetraacetic acid or EDTA abolishes phosphatidylinositol degradation. However, in the presence of added Ca2+, other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) are also degraded but by a phospholipase A2 activity. This activity generates the respective lyso-derivatives as well as various [14C]arachidonate metabolites. The phospholipase A2 activity is further enhanced by increasing the pH (7.5 to 9.5), a condition which severely suppresses the phospholipase C activity. Most of the platelet phospholipase A2 activity is associated with the particulate fractions of the cell, while the phospholipase C activity appears to be almost completely restricted to the soluble fraction. Deoxycholate treatment of the particulate fractions results in cleavage by phospholipase A2 of phosphatidylcholine and phosphatidylethanolamine but not of phosphatidylinositol. The preferred substrates for platelet phospholipase A2 appear to be phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine, while phosphatidylinositol seems to be degraded nearly exclusively by phospholipase C.
Topics: Animals; Arachidonic Acids; Blood Platelets; Calcium; Diglycerides; Horses; Hydrogen-Ion Concentration; Kinetics; Phospholipases; Phospholipases A; Phospholipases A2; Substrate Specificity; Type C Phospholipases
PubMed: 7430120
DOI: No ID Found -
FEBS Letters Oct 2002Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH... (Review)
Review
Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.
Topics: Animals; Binding Sites; Blood Proteins; Catalysis; Humans; Isoenzymes; Models, Molecular; Phospholipase C beta; Phospholipase C delta; Phosphoproteins; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Type C Phospholipases
PubMed: 12401198
DOI: 10.1016/s0014-5793(02)03411-7 -
Annals of the Rheumatic Diseases Nov 1989
Topics: Arthritis, Rheumatoid; Humans; Inflammation; Phospholipases; Phospholipases A; Phospholipases A2; Synovial Fluid
PubMed: 2596887
DOI: 10.1136/ard.48.11.962-b -
Journal of Medicinal Chemistry Sep 2020The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca-independent production of...
The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca-independent production of -acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive -acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the α-ketoamide series using activity-based protein profiling. This led to the identification of , a nanomolar potent inhibitor for the PLAAT family members. reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, may help to dissect the physiological role of the PLAATs.
Topics: Acyltransferases; Amides; Enzyme Inhibitors; Hep G2 Cells; Humans; Models, Molecular; Phospholipases; Protein Conformation; Structure-Activity Relationship
PubMed: 32787138
DOI: 10.1021/acs.jmedchem.0c00522 -
Journal of Lipid Research Mar 1981Current assays of phospholipase A1 (EC 3.1.1.32) and monoacylglycerol lipase (EC 3.1.1.23) activities in tissues are discontinuous, laborious, and expensive. Some...
Current assays of phospholipase A1 (EC 3.1.1.32) and monoacylglycerol lipase (EC 3.1.1.23) activities in tissues are discontinuous, laborious, and expensive. Some spectrophotometric substrates were synthesized to alleviate this problem. Thioester analogs of phosphatidylcholine and phosphatidylethanolamine. rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-3-phosphoethanolamine-1-mercapto-2,3-propanediol, were synthesized from the diacylglycerol analog, rac-1,2-S,O--didecanoyl-1-mercapto-2,3-propanediol. The latter was prepared from triacylmercaptoglycerol by lipolysis and separation by chromatography on silica gel. Monoacylglycerol thioester analogs, 1-S-hexadecanoyl-1-mercapto-2,3-propanediol and 1-S-decanoyl-1-mercapto-2,3-propanediol, were synthesized by selective acylation of mercaptoglycerol. All of the substrates were hydrolyzed by Rhizopus delemar lipase to release sulfhydryl groups reactive towards 4,4'-dithiobispyridine. The hydrolysis could be followed continuously in a spectrophotomer with 0.1 absorbance unit corresponding to 5 nmol product. The structure and isomeric purity of the phospholipid analogs were verified by their behavior on thin-layer chromatography, elemental analyses, infrared spectra, and by the specificity of the colorimetric reaction with lipolytic enzymes. Whereas phospholipase A1 activity hydrolyzed both phospholipid analogs to release the theoretical amount of free thiol, neither phospolipases C nor A2 promoted thio release. The pH optimum, heat stability, and Ca2+ ion dependency were determined for the hydrolysis of each substrate by R. delemar lipase. The results indicate that the phospholipase A1 and monoacylglycerol lipase activities in R. delemar lipase are due to separate enzymes, and that these enzyme specific assays will be of general utility for enzyme characterization and purification studies. These substrates are useful for sensitive, convenient, and specific spectrophotometric assays for phospholipase A1 and monoacylglycerol lipase over the pH range 3 to 8.
Topics: Calcium; Carboxylic Ester Hydrolases; Kinetics; Monoacylglycerol Lipases; Phospholipases; Phospholipases A; Phospholipases A1; Phospholipids; Rhizopus; Spectrophotometry, Infrared; Substrate Specificity
PubMed: 7240974
DOI: No ID Found -
Journal of Lipid Research Dec 1984Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester... (Comparative Study)
Comparative Study Review
Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester substrate analogs as well as omega-nitrophenyl derivatives of the corresponding lipids. The most promising results are obtained with the thioester substrate analogs. Mono- and diacylglycerol lipases are assayed by using rac-1-S-decanoyl-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-1-mercapto-2,3-propanediol, respectively. Phospholipases A1 and A2 are determined by using rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and 2-hexadecanoylthio-1-ethyl-phosphocholine, respectively. Lysophospholipases are measured by using 2-hexadecanoylthio-1-ethyl-phosphocholine. Phospholipase C is assayed with rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol. Thioester substrate analog assay procedures are more rapid, sensitive, convenient, continuous, and less expensive than the classical radiochemical techniques.
Topics: Lipase; Lipoprotein Lipase; Lysophospholipase; Monoacylglycerol Lipases; Phospholipases; Phospholipases A; Spectrophotometry; Sulfides; Type C Phospholipases
PubMed: 6397560
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1979Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible... (Comparative Study)
Comparative Study
Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.
Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.
Topics: Amino Acids; Animals; Bacteria; Blood Bactericidal Activity; Blood Proteins; Dactinomycin; Humans; Molecular Weight; Neutrophils; Permeability; Phospholipases; Phospholipases A; Phospholipases A2; Rabbits; Species Specificity
PubMed: 500619
DOI: No ID Found -
The Biochemical Journal Nov 1981Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a...
Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.
Topics: Animals; Ascitic Fluid; Cells, Cultured; Culture Media; Guinea Pigs; Hydrogen-Ion Concentration; Macrophages; Male; Mice; Mice, Inbred Strains; Phospholipases; Phospholipases A; Phospholipases A2; Zymosan
PubMed: 7340844
DOI: 10.1042/bj2000441 -
Gut Nov 1987The influence of ischaemia and revascularisation on lipid peroxidation and phospholipid metabolism in the rat small intestinal mucosa was investigated. Two hours of...
The influence of ischaemia and revascularisation on lipid peroxidation and phospholipid metabolism in the rat small intestinal mucosa was investigated. Two hours of total ischaemia followed by five minutes of revascularisation caused not only accumulation of malondialdehyde in the mucosa, but also increased activity of phospholipase A2, decreased activity of lysophospholipase, and increased ratio between lysophosphatidylcholine and phosphatidylcholine. Pretreatment with the phospholipase A2 inhibitor, quinacrine, prevented the increases in mucosal phospholipase A2 activity and lysophosphatidylcholine/phosphatidylcholine ratio after ischaemia and morphological examinations revealed that the mucosa was then also protected against ischaemic injury. These findings point to the possibility that activation of phospholipase A2 and accumulation of lysophosphoglycerides could be involved in mediating the mucosal injury caused by small intestinal ischaemia.
Topics: Animals; Female; Intestinal Mucosa; Intestine, Small; Ischemia; Lysophospholipase; Malondialdehyde; Phospholipases; Phospholipases A; Phospholipases A2; Quinacrine; Rats; Rats, Inbred Strains
PubMed: 3428670
DOI: 10.1136/gut.28.11.1445 -
Journal of Biochemistry Feb 1979The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were...
The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.
Topics: Amino Acids; Calcium; Elapid Venoms; Fatty Acids; Humans; Isoenzymes; Kinetics; Molecular Weight; Phospholipases; Spectrophotometry, Ultraviolet
PubMed: 422537
DOI: 10.1093/oxfordjournals.jbchem.a132344