Acid phospholipase A1 in liver — A brief survey

Summary Acid phospholipase A₁ activity in liver (rat, human) is predominantly localized in lysosomes. A minor proportion (less than 3% of the total activity) is also present in the Golgi apparatus and the endoplasmic reticulum, presumably due to enzymatically active precursors of the corresponding lysosomal enzyme. Lysosomal phospholipase A₁ is the most important enzyme initiating the intralysosomal catabolism of diacylphosphoglycerides. It has been purified 50,600-fold, with a yield of about 26%. The enzyme prefers phosphatidylethanolamine as a substrate, which at 200 µM and pH 4.5, is hydrolysed at a rate of approximately 8.2 U/mg. Lysosomal phospholipase A₁ is a glycoprotein of about 29 kDa with an isoelectric point of pH 5.3. Unspecific extralysosomal endogenous inhibitors of this enzyme are pH range, inorganic cations, and various proteins. Divalent cations are more potent inhibitors than monovalent ones. Most endogenous intra- and extracellular proteins inhibit the enzyme, the cationic species exhibiting high inhibitory potencies, glycoproteins only little. Inhibitory proteins act through their binding to the substrate. Lysosomal phospholipase A₁ seems to be an important target in drug-induced lipidosis. This lipid storage disease is caused by various cationic amphiphilic drugs that are trapped intralysosomally by protonation. In lysosomes such compounds raise the pH, interact with the polar lipids to be degraded and the lysosomal lipolytic enzymes, such as phospholipase A₁. These mechanisms result in impaired intralysosomal phospholipid degradation and hence in intralysosomal phospholipid accumulation.Dedicated to Professor Walther Vogt on the occasion of his 70th birthday     

In vitro inhibition of phospholipase A2 by gabexate mesilate,camostate, and aprotinine

Summary Gabexate mesilate (FOY, ethyl-p-(6-guanidino-hexanoyl-oxy)-benzoate-methansulfonate), camostate (N,N-dimethyl-carb-amoylmethyl-4-(guanidinobenzoyloxy)-phenyl-acetate) methansulfonate and aprotinine (Trasylol) were tested for possible inhibition of phospholipase A₂. Gabexate mesilate at a concentration of 5×10^–4 mol/l and camostate at a concentration of 10^–3 mol/l caused a 50% reduction in enzyme activity. There was almost no inhibition by aprotinine at clinical doses; 40 million KIU/l were necessary to reduce phospholipase A₂ activity by 20%. From the therapeutic dose (4,000 mg/day per i.v. infusion) and the half-life of gabexate mesilate in blood circulation (1 min) it can be calculated that the in vitro concentration of gabexate mesilate is only 10^–6 to 10^–7 mol/l. Under these conditions gabexate mesilate cannot diminish the in vivo enzyme activity of phospholipase A₂.     

Inhibition of porcine pancreas phospholipase A2 activation by gabexate mesilate

Summary We investigated the effect of gabexate mesilate on the catalytic activity of phospholipase A₂ in homogenized porcine pancreatic tissue. Gabexate mesilate is a potent inhibitor of serine proteases. There is no direct inhibition of phospholipase A₂ catalytic activity in concentrations up to 6 mmol/l. Preincubation of homogenized pancreatic tissue with gabexate mesilate leads to a reduction of phospholipase A₂ activity even in concentrations as low as 6 µmol/l. The activation of purified porcine prophospholipase A₂ added to pancreatic tissue can be completely inhibited. Thus, gabexate mesilate might influence the activation of phospholipase A₂ administered in therapeutic concentrations in inflamed pancreatic tissue.Abbreviations PLA₂ Phospholipase A₂ - ProPLA₂ Prophospholipase A₂     

Serum phospholipase A2 in intensive care patients with peritonitis,multiple injury,and necrotizing pancreatitis

Summary To study the source and role of circulating phospholipase A₂ (PLA₂) catalytic activity we monitored the serum from patients with necrotizing pancreatitis (n=8), diffuse peritonitis (n=6), and multiple injuries (n=11). Immunoreactive PLA₂ serum protein concentration was analysed using a fluoroimmunoassay based on an antibody against human pancreatic PLA₂. Serum PLA₂ catalytic activity was analysed using a radiochemical method based on a substrate with tritiated palmitic acid in beta position. In necrotizing pancreatitis immunoreactive PLA₂ and PLA₂ catalytic activity both increased. Obviously, in necrotizing pancreatitis the major part of serum catalytic activity stems from the pancreas. In patients with diffuse peritonitis and multiple injuries, as a rule, immunoreactive phospholipase A₂ serum concentration appears to be within the normal range. In contrast, in these patients we demonstrated high serum catalytic PLA₂ activity comparable to that in necrotizing pancreatitis. The source of catalytic PLA₂ activity in peritonitis and multiple injuries seems not to be the pancreas. There was a correlation between pulmonary insufficiency and serum PLA₂ catalytic activity in patients with necrotizing pancreatitis, peritonitis, and multiple injuries.     

Properties of lysophospholipase in Mycobacterium leprae

Lysophospholipids are key intermediates in the metabolism of phospholipids. Cytoplasmic membranes of both eukaryotes and prokaryotes are made of phospholipid bilayers. Phospholipases are activated during phagocytosis. Lysophospholipids generated by phospholipase A₂ or A₁ degrade cell membranes and can cause cell lysis. An active lysophospholipase, that hydrolyzes lysophospholipids, was detected by the radioisotope technique in Mycobacterium leprae. About two-thirds of the enzyme was particulate and one-third cytoplasmic. Optimum activity was at 37 °C, and at pH 6.0. Temperatures above 70 °C completely inactivated the enzyme. The compound AACOCF3, a trifluromethylketone analog of arachiodonic acid, inhibited the activity; the inhibition appeared to be of the uncompetetive type. The K_m of the enzyme was 2.5 × 10^?4 M, suggesting a fairly strong affinity for the substrate. Lysophospholipids have been shown to be microbicidal to invading organisms. Possession of lysophospholipase by M. leprae is apparently one of the methods by which the bacilli overcome the defense mechanisms of the host.     

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)₂D₃, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)₂D₃ treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (R_a 0.54 and 4.92 μm) in the presence of control media or media containing 1,25-(OH)₂D₃ or 1,25-(OH)₂D₃ plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A₂ inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)₂D₃ inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)₂D₃ restored cell number to that seen in the control cultures. Inhibition of phospholipase A₂ in the presence of 1,25-(OH)₂D₃ caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)₂D₃ on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)₂D₃ treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)₂D₃ displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A₂ also inhibited the 1,25-(OH)₂D₃-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)₂D₃ effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)₂D₃ mediate their effects through phospholipase A₂, which catalyzes one of the rate-limiting steps in prostaglandin E₂ production. Further downstream, prostaglandin E₂ activates protein kinase A.     

Effects of hormones on phospholipase A2 activity in rat skin

We studied the effects of hydrocortisone, progesterone, estradiol, and testosterone on phospholipase A₂ activity in plasma and lysosomal membranes of rat dermis and epidermis. The dermal enzyme was more sensitive to estradiol, while epidermal to testosterone. The effects of progesterone and hydrocortisone were mediated by lipocortin-like protein, while estradiol and testosterone directly by modulated phospholipase A₂ activity Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 70–72, January, 2000     

A rapid phospholipase A2 bioassay using14C-oleate-labelledE. coli bacterias

Summary Two methods of phospholipase A₂ determination using¹⁴C-labelledE. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved¹⁴C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A₂ regarding pH and Ca²⁺ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A₂ levels, but that the filter method is superior in terms of sensitivity and workload.Abbreviations PLA₂ phospholipase A₂ - BSA bovine serum albumin - (F)FA (free) fatty acid - TLC thin-layer chromatography     

Suramin inhibits macrophage activation by human group IIA phospholipase A2, but does not affect bactericidal activity of the enzyme

Objective:  Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A₂ (hsPLA₂GIIA). Materials and Methods:  The hsPLA₂GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA₂ GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. Results:  The hydrolytic activity of the hsPLA₂ GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA₂ GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA₂ GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 μM. Conclusions:  These results demonstrate that suramin selectively inhibits the activity of the hsPLA₂ GIIA against macrophages, whilst leaving the anti-bacterial function unchanged. Received 26 June 2008; returned for revision 8 August 2008; received from final revision 11 September 2008; accepted by J.Skotnicki 8 October 2008     

Role of Phospholipase A2 in Activation of Isolated Cardiomyocyte Respiration in Postinfarction Cardiosclerosis

The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A₂ in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A₂ in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A₂. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 631–633, December, 2008     

Wechselwirkungen der Aldehyddehdyrogenase aus Acinetobacter calcoaceticus mit Membranlipiden II. Rekonstitution in künstlichen Membranvesikeln

Purified aldehyde dehydrogenase (NADP⁺-dependent) of intracytoplasmic membranes of Acinetobacter calcoaceticus could be incorporated from micelles formed during the purification procedure into liposomal membranes. Both the cholate dilution method and the ultrasonication method were suitable to produce enzyme liposomes. In unilamellar liposomes produced by phosphatidyl choline, the enzyme activity decreased to 1% (or less) of the original activity. In contrast, about 10% of the original activity could be preserved in unilamellar liposomes prepared from bacterial phospholipids. The destruction of the enzyme liposomes induced by detergents (lauroyl sarcosinate) was followed by measuring the wavelength dependence of turbidity, which allowed us to draw conclusions on size and stability of the particles in the suspension. In addition these measurements demonstrated that decanal and NADP⁺ did not destroy the liposomal structure at concentrations necessary for the determination of enzyme activity. The liposomal enzyme was inactivated to a lesser degree by proteinase K than the micellar enzyme. Both phospholipase A₂ and D inactivated the enzyme incorporated into the liposomal membranes to about 50%. After treatment with phospholipase A₂, the enzyme could be reactivated by bacterial phospholipids. After treatment with phospholipase D, no reactivation was possible by bacterial phospholipids.     

A radiochemical test for phospholipase A2 catalytic activity

Summary A position-specifically labelled phosphatidylcholine is the substrate for the selective determination of Phospholipase A₂ in serum, ascites and tissue samples. Optimal reaction conditions and simplifications of handling are discussed.A control group of human serum samples ranged up to 2.1 U/l. The maximum serum activity in samples of patients with acute pancreatitis was 126 U/l. In human ascites activities up to 380 U/l were measured. The method described here turned out to be a practicable instrument for the determination of phospholipase A₂ activity using only commercially available reagents.Abbreviation PlA₂ Phospholipase A₂     

Zinc (Zn2+) binds to and stimulates the activity of group i but not group II phospholipase A2

Phospholipase A₂ plays an important part in the generation of inflammatory lipid mediators and so it is of major interest to understand functional distinctions between structurally similar forms of phospholipase A₂. In the present study, the influence of zinc (Zn²⁺) on the activity of group I and group II phospholipase A₂ was examined in vitro. It appeared that Zn²⁺ (0.04–1×10^–3 M) increased group I phospholipase A₂ activity from porcine pancreas and rat lung whereas the activity of group II phospholipase A₂ fromCrotalus atrox andVipera russelli was unaffected. The presence of Cd²⁺ of Hg²⁺ (0.8–5×10^–3 M) also increased group I pancreatic phospholipase A₂ activity while no augmentation was found with Cr²⁺, Fe²⁺ or Mg²⁺. The selective stimulation of group I phospholipase A₂ by Zn²⁺ corresponded to a binding of these phospholipases A₂ to a zinc-affinity column, while group II phospholipase A₂ was not bound. Furthermore, the PLA₂ activity in bronchoalveolar lavage fluid from rat was stimulated by Zn²⁺. These results indicate that Zn²⁺ binds to and increases the activity of group I, but not group II phospholipase A₂. This difference in Zn²⁺-binding may be used to discriminate between group I and group II phospholipase A₂ and to separate the enzymes from each other in complex biological materials. The possibility that activation of group 1 phospholipase A₂ in the lung is important in zinc-induced metal fume fever is implied.     

In vivo inhibition of phospholipaseA 2 in rat lung by an β-adrenergic agonist

Phospholipase A₂ is the key enzyme for the release of arachidonic acid derived mediators via cycloxygenase or lipoxygenase pathways in inflammation and immunological processes. Activity of microsomal phospholipase A₂ in rat lung can be inhibited byin vivo application of the ß-adrenergic drug fenoterol both in control and diseased animals.     

Groups I,II and III extracellular phospholipases A2: Selective inhibition of group II enzymes by indomethacin but not other NSAIDs

The three types (groups I, II and III) of stable extracellular 14 kDa phospholipase A₂ enzymes differ in their primary amino acid sequences and their properties. It may thus be possible to design low-molecular weight inhibitors targeted to the secretory form of mammalian PLA₂. this enzyme has been implicated in inflammatory disorders. We have studied the inhibition of four distinct PLA₂ enzymes by a range of NSAIDs, using³H-oleate release from prelabelled membranes ofE. coli for assay. The enzymes used were cobra venom PLA₂ (Naja naja, a group I enzyme), bee venom PLA₂ (Apis mellifera, group III), recombinant human synovial PLA₂ (group II) and rat peritoneal PLA₂ (group II). Under the conditions of the³H-oleateE. coli assay, 1 mM concentrations of aspirin, sodium salicylate, paracetamol (acetaminophen), oxphenbutazone, ibuprofen, flurbiprofen and nabumetone failed to inhibit significantly any of the four enzymes. However, indomethacin inhibited all four enzymes, although effects were greatest on the two group II enzymes (rat peritoneal and human synovial PLA₂). Approximate IC₅₀ values were 28 and 35 M, respectively. Inhibition by indomethacin was not time dependent and was greater at micromolar rather than millimolar levels of calcium. We conclude that indomethacin but not the other tested classes of NSAID inhibits the group II PLA₂ enzyme in a selective manner and suggest that this may be relevant both to its clinical spectrum and to the design of novel pharmaceutical leads.     

Immunocytochemical evidence of phospholipase A2 in pancreatic tumors — Diagnostic values

Summary Phospholipase A₂ is an enzyme which is produced in acinar cells, and persists even in regressive states of chronic pancreatitis, when the production of other enzymes diminishes. We therefore tested this enzyme as a marker of acinar descent in various pancreatic tumors. This enzyme could be seen in 50% of the acinar-cell carcinomas, in 60% of solid and papillary pancreatic tumors, and in 50% of microglandular carcinomas. Ductal cancers and isletcell cancers were negative. In contrast to other markers of acinar matrix (amylase, antitrypsin), phospholipase A₂ gave fewer false-positive or false-negative results.Supported by a grant of the J. and F. Marohn Stiftung at the University of Erlangen/Nürnberg     

Lysolecithin concentration in pancreatic tissue during therapy with phospholipase A2-inhibitors in acute necrotizing pancreatitis

Summary Four hours after hemorrhagic necrotizing pancreatitis was induced in experimental animals the concentration of lysolecithin in pancreatic tissue rose (p=0.0025). Parenteral administration of unspecific inhibitors of phospholipase A₂, such as chlorpromazine, gabexat mesilat and CaNa₂ EDTA, can neither inhibit elevation of the enzyme nor influence the course of the disease.     

The phospholipase A2 from human platelets

Studies on a purified phospholipase A₂ (PLA₂) from human platelets show that the enzyme, which is copurified with the plasma membrane fraction, has a MW of 50 K Dalton, requires Ca⁺⁺, and has a pH optimum of 9.4. Under optimal conditons, PLA₂ activity corresponds to at least 13 nmol/min/10⁹ platelets. Unsaturated PL are preferred substrates and the enzyme is considerably more active on the aggregated form of the substrate than on the monomers. The specific activity is markedly affected by the quality of the interface, showing variations of more than 10-fold between different substrate forms. In the absence of detergents, a 4-fold increase in rate is observed when both products are present. Maximal rates are obtained at 20 mole percent of products to substrate. 1,2-Diglyceride and phosphatidic acid stimulate the hydrolysis of PC by the purified enzyme, however, in these forms of the substrate, neither of them are hydrolyzed. Activation of this enzyme by some intermediate of the phospholipase C pathway might play a role in the stimulus-linked release of platelet arachidonic acid.To whom correspondence should vbe addressed.     

Occurrence of phospholipase A2 and lysophospholipase in a gastric H,K-ATPase-containing membrane fraction,and the formation of lysophosphatidylcholine in stimulated pig parietal cells

A membrane fraction containing -ATPase (EC 3.6.1.36) was prepared from pig gastric mucosa and found to contain phospholipase A, (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5) activities. Washing the membranes decreased their protein content by 25%. Recovery profiles of -ATPase, phospholipase A, and lysophospholipase were similar for membranes washed either with water or with 0.15 or 1.5 m KCI. Nearly identical distribution profiles were obtained for the three enzyme activities after centrifugation of washed vesicle membranes on a linear sucrose gradient. The phospholipase A₂ activity was stimulated by calcium and increased further in the presence of calmodulin. The amount of cellular radioactively labelled lysophosphatidylcholine was doubled upon cholinergic stimulation of isolated parietal cells prelabelled with [³H]glycerol or ³²P₁. The liberated lyso(³²P]phosphatidylcholine had its acyl chain in the sn-1 position, which implies an activation of a phospholipase A₂. These findings indicate that secretagogues which increase the cytosolic Ca²⁺ concentration, i.e. acetylcholine, histamine and gastrin, may activate a phospholipase A₂ in the parietal cell.     

On the present and future role of Lp-PLA2 in atherosclerosis-related cardiovascular risk prediction and management

Circulating concentration and activity of secretory phospholipase A₂ (sPLA₂) and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) have been proven as biomarkers of increased risk of atherosclerosis-related cardiovascular disease (ASCVD). Lp-PLA₂ might be part of the atherosclerotic process and may contribute to plaque destabilisation through inflammatory activity within atherosclerotic lesions. However, all attempts to translate the inhibition of phospholipase into clinically beneficial ASCVD risk reduction, including in randomised studies, by either non-specific inhibition of sPLA₂ (by varespladib) or specific Lp-PLA₂ inhibition by darapladib, unexpectedly failed. This gives us a strong imperative to continue research aimed at a better understanding of how Lp-PLA₂ and sPLA₂ regulate vascular inflammation and atherosclerotic plaque development. From the clinical viewpoint there is a need to establish and validate the existing and emerging novel anti-inflammatory therapeutic strategies to fight against ASCVD development, by using potentially better animal models and differently designed clinical trials in humans.