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PubMed1984

Improvement of the anoxia-induced mitochondrial dysfunction by membrane modulation.

The mitochondrial dysfunction induced by anoxia in vitro was improved with chlorpromazine, cepharanthine, bromophenacyl bromide, and mepacrine without affecting phospholipid or adenine nucleotide metabolisms. The drugs inhibited lipid peroxidation by Fe2+, mitochondrial disruption by Ca2+, and membrane perturbation by lysolecithin, and retained the activity to control H+ permeability across mitochondrial membranes. The drugs appeared to preserve the functions by acting to suppress the development of membrane deterioration which may have resided in the deenergization of mitochondria in the absence of oxygen.

Acetophenones↗
PubMed1987

Diacylglycerol breakdown in plasma membranes of bovine chromaffin cells is a two-step mechanism mediated by a diacylglycerol lipase and a monoacylglycerol lipase.

The recently identified diacylglycerol lipase activity in membranes of chromaffin cells from bovine adrenal medulla [24] is now shown to consist of two enzymes working in series. First the predominantly saturated fatty acid in the sn-1-position is split by a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.34). Subsequently the resulting sn-2-monoacylglycerol is split by a monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) which prefers sn-2-arachidonoyl-monoacylglycerol to sn-2-palmitoyl-monoacylglycerol. At pH 4.0 only the diacylglycerol lipase is active, whereas the monoacylglycerol lipase is irreversibly inactivated. At pH 6.0 both enzymes are active. Pretreatment of the membranes at pH 10 leads to the selective inactivation of the diacylglycerol lipase. Both enzymes are Ca2+- and calmodulin-independent and both are partially inhibited by p-bromophenacyl bromide, however, only at relatively high concentrations of the inhibitor. Chlorpromazine inhibits the diacylglycerol lipase to about the same extent as p-bromophenacyl bromide but the monoacylglycerol lipase is less sensitive. The specific diacylglycerol lipase inhibitor RHC 80267 (1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane) only interacts with the first step, i.e. the diacylglycerol lipase.

Acetophenones↗
PubMed1977

Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.

Acetophenones↗
PubMed1977

New formamidase having substrate specificity for o-formylaminoacetophenone in pig liver.

The supernatant (13 000 x g, 20 min) of pig liver homogenate was filtered with Sephadex G-200 and formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9)activity in each fraction was measured. When formylkynurenine was used as substrate, two peaks of formamidase activity were observed but, with formylaminoacetophenone as substrate, only one peak was observed. Formamidase in the lower molecular weight fraction is known as kynurenine formamidase (FA I), formamidase found here in the higher molecular weight fraction has not been previously reported. This form, designated FA II has been purified about 160-fold from pig liver. The formamidase obtained has substrate specificity for o-formylaminoacetophenone only and could not hydrolyze formylkynurenine. The optimal pH was 8.5 and the Km for o-formylaminoacetophenone was 1.66-10(-3) M. This formamidase was considered to be a new enzyme and was different from FA I in molecular weight and substrate specificity. This new formamidase was present in pig, rabbit and guinea pig liver and not present in rat or mouse liver.

Acetophenones↗
PubMed1980

Chemical modification of the histidine residue in basic phospholipase A2 from the venom of Naja nigricollis.

Phospholipase A2 from Naja nigricollis venom was separated into three fractions by chromatography on a column of CM-Sephadex C-25. The pI values of fractions CMS-5, CMS-6 and CMS-9 were determined to be 7.6, 8.3 and 10.6, respectively. Fraction CMS-9 was further purified on a DEAE-Sephacel column and the homogeneity was verified. The specific activity of CMS-9 was found to be 1300 units per mg and lethal toxicity 0.3 mg per kg mouse. The most basic and toxic fraction, CMS-9, was subjected to chemical modification with p-bromophenzcyl bromide. The enzyme lost both the enzyme activity and lethal toxicity, however, the antigenicity remained unchanged. Although both 8-anilinonaphthalenesulfonate and Ca2+ showed pronounced protection on the inactivation process, the mechanism of 8-anilinonaphthalene-sulfonate protection is different from that of Ca2+. Amino acid analysis showed that only one (His-47) out of three histidine residues was modified. Although both native and His-modified CMS-9 were perturbed by the presence of Ca2+, the modified enzyme lost the characteristic tryptophan blue shift suggesting that the modified enzyme is unable to exert a charge effect upon Ca2+ binding in the vicinity of the tryptophan group. Scatchard plots revealed only one type of binding sites for 8-anilinonaphthalenesulfonate in the presence of Ca2+. On the other hand, the modified enzyme lost the ability to bind 8-anilinonaphthalene. It is suggested tentatively that the hydrophobic pocket in which 8-anilinonaphthalenesulfonate is bound may be the site of the enzyme that interacts with phospholipid.

Acetophenones↗
PubMed1982

Studies on the incorporation of [1-14C]arachidonic acid into glycerolipids and its conversion into prostaglandins by rabbit iris. Effects of anti-inflammatory drugs and phospholipase A2 inhibitors.

The effects of the anti-inflammatory drugs, indomethacin and aspirin, and the phospholipase A2 inhibitors, p-bromophenacyl bromide and mepacrine, on the in vitro metabolism of [1-14C]arachidonic acid by rabbit iris smooth muscle and iris microsomes were investigated. The incorporation of arachidonate into glycerolipids and its conversion into prostaglandins were rapid and time-dependent. About 65% of the total radioactivity was recovered in triacylglycerol, followed by that in phosphatidylcholine (20%), diacylglycerol (6%), phosphatidylethanolamine (5%) and phosphatidylinositol (3%), respectively. Time-course studies on arachidonate release from glycerolipids of prelabelled tissue showed that triacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major source for arachidonate in prostaglandin synthesis in this tissue. Arachidonate release from glycerolipids was not blocked by indomethacin and the effects of the phospholipase A2 inhibitors were nonspecific. p-Bromophenacyl bromide inhibited the labelling of glycerolipids in a dose-dependent manner. Mepacrine stimulated the labelling of phosphatidic acid, phosphatidylinositol and diacylglycerol, and inhibited that of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol. At concentrations under 0.25 mM it stimulated prostaglandin synthesis in microsomes and at concentrations over 0.25 mM it inhibited their synthesis in both muscle and microsomes. Indomethacin and aspirin moderately increased the labelling of glycerolipids; however, both drugs inhibited prostaglandin synthesis by iris and iris microsomes in a dose-dependent manner. Possible explanations for mechanisms underlying these effects were presented. It is concluded that the phospholipase A2 inhibitors and the anti-inflammatory drugs exert profound effects on the incorporation of [1-14C]arachidonate into glycerolipids of the rabbit iris and on its conversion into prostaglandins by both iris and iris microsomes.

Acetophenones↗
PubMed1983

Surface-active phospholipase A2 in mouse spermatozoa.

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.

Acetophenones↗
PubMed1983

Mechanisms involved in the synthesis of disaturated phosphatidylcholine by alveolar type II cells isolated from adult rat lung.

1. Alveolar type II cells isolated from adult rat lung incorporated radioactively labelled palmitate predominantly into the 2-position of disaturated phosphatidylcholine. In disaturated diacylglycerol, however, the radioactivity was almost equally distributed between the 1- and 2-positions. 2. Exposure of isolated type II cells to the phospholipase A2 inhibitors 4-bromophenacylbromide or quinacrine dihydrochloride led to a decreased synthesis of total phosphatidylcholines from various labelled precursors. Interestingly, it also led to an increased degree of unsaturation of the phosphatidylcholines synthesized by the cells. 3. Incubation of type II cell sonicates with radioactively labelled CDPcholine resulted in the formation of labelled phosphatidylcholine; 56% of this phosphatidylcholine appeared to be disaturated. In similar experiments with homogenates from whole lung, 20% of the synthesized phosphatidylcholine was disaturated. 4. These results suggest that both direct synthesis de novo and remodeling of 1-saturated-2-unsaturated phosphatidylcholines contribute to the biosynthesis of disaturated phosphatidylcholine in isolated alveolar type II cells.

Acetophenones↗
PubMed1984

Nonspecific inhibition of enzymes by p-bromophenacyl bromide. Inhibition of human platelet phospholipase C and modification of sulfhydryl groups.

This study demonstrates that p-bromophenacyl bromide irreversibly inhibits, in a time- and dose-dependent manner, yeast alcohol dehydrogenase, bovine pancreatic alpha-chymotrypsin, human platelet phosphatidylinositol (PI)-specific phospholipase C, in addition to the neutral-active and calcium-dependent phospholipase A2 of human platelets. The PI-specific phospholipase C has maximal activity at pH 5,5 is calcium-dependent, and is strongly inhibited by sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and methylmethane thiosulfonate . Increasing concentrations of DTNB produced concomitant inhibition of phospholipase C activity and titration of sulfhydryl groups. In contrast, human platelet phospholipase A2 activity was unaffected by concentrations of DTNB that titrated sulfhydryl groups, and completely inhibited PI-specific phospholipase C activity. Treatment of cysteine with p-bromophenacyl bromide resulted in modification of the amino acid as demonstrated by paper chromatography, and loss of titratable sulfhydryl groups. These data show that p-bromophenacyl bromide inhibits a wide spectrum of enzymatic activities including PI-specific phospholipase C. This reagent modifies amino acid residues other than active-site histidines and therefore has a broader reactivity than previously considered. Thus, it should not be used as a selective inhibitor of enzymes in crude cellular experiments.

Acetophenones↗
PubMed1985

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes.

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.

Acetophenones↗
PubMed1986

Identification of a calcium-independent phospholipase A2 in rat lung cytosol and differentiation from acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether).

The 100 000 X g supernatant of total rat lung homogenate was found to contain at least three phospholipase A2-type activities. Gel filtration separated a low molecular weight and Ca2+-requiring phospholipase A2 from Ca2+-independent acylhydrolase peak with an apparent higher molecular weight. Upon DEAE-cellulose chromatography this fraction was separated into a Ca2+-independent acylhydrolase and a Ca2+-independent platelet-activating factor-acetylhydrolase with no apparent overlap in acyl chain length specificity. The long-chain acylhydrolase was shown to exhibit specificity for the ester bond at the sn-2-position. Ca2+-independent phospholipase A2 activity was inhibited by p-bromophenacylbromide and was resistant to diisopropylfluorophosphate. In contrast, the Ca2+-independent acetylhydrolase activity was inhibited by diisopropylfluorophosphate but was unaffected by p-bromophenacylbromide.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1987

Contamination of commercial phosphatidylcholine by phospholipase A.

During the course of a study involving the assay of a membrane-bound phospholipase A2 it was observed that a commercial preparation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine used as substrate had intrinsic lipolytic activity at pH 8.5. Further investigation revealed a Ca2+-dependent phospholipase A largely susceptible to treatment by the alkylating reagent p-bromophenacyl bromide or by heat (15 min at 120 degrees C). Complete separation of enzyme and phospholipid could be achieved by thin-layer chromatography. Such a contamination was not observed in a chemically identical phosphatidylcholine obtained from a different supplier. These observations may be relevant to investigators using commercial preparations of phospholipids in a variety of studies, including intracellular phospholipase A2 determination.

Acetophenones↗
PubMed1988

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.

Acetophenones↗