Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute f1-2 regiospecificity for diradyl glycerophospholipids

Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 mumol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine greater than alkyl-ether choline glycerophospholipid greater than phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.     

Purification, characterization and bactericidal activities of basic phospholipase A2 from the venom of Agkistrodon halys (Chinese pallas)

Agkistrodon snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this study, we used reverse-phase HPLC to purify PLA(2) from the venom of Agkistrodon halys. The enzyme named as AgkTx-II, a basic Asp49 PLA(2), has a molecular masses of 13,869.05. The amino acid sequence and molecular mass of AgkTx-II was identical to those of an Asp49 basic myotoxic PLA(2) previously isolated from this venom. Antibacterial activities were tested by susceptibility and broth-dilution assays. AgkTx-II exerted a potent antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. The MIC values of AgkTx-II ranged between 85 and 2.76muM and was most effective against S. aureus, P. vulgaris, P. mirabilis (MIC of 21.25muM) and B. pseudomallei (MIC of 10.25muM). This AgkTx-II rapidly killed S. aureus, P. vulgaris and B. pseudomallei in a dose-dependent manner. The effect of the AgkTx-II on bacterial membranes was evaluated by scanning and transmission electron microscopy. AgkTx-II caused morphological alterations apparent on their cellular surfaces, suggesting a killing mechanism based on membrane permeabilization and damage. Cytotoxicity was measured by XTT tetrazolium (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and lactate dehydrogenase (LDH) assays using U-937 cells (monocytes). The AgkTx-II did not affect cell viability up to 500muM concentrations but cell death was evident at 1000muM concentration after 24 and 48h. Furthermore, the repeated exposure of AgkTx-II (2-14muM) treated mice showed different tissue alterations, mainly at the brain and kidney; the toxicological potential of AgkTx-II remains to be elucidated. The AgkTx-II exhibits no hemolytic action even at high doses (10-100muM) in human erythrocytes. However, the AgkTx-II is believed to exert its bactericidal effect by permeabilizing the bacterial membrane by forming pores. In addition, the basic PLA(2) AgkTx-II displays a bactericidal effect, which may be either dependent or independent of catalysis.     

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.     

Purification and properties of phospholipase A2 from the venom of scorpion, (Heterometrus fulvipes)

Phospholipase A2 was purified about 78 fold from the venom of scorpion Heterometrus fulvipes. The molecular weight of the enzyme was found to be 16,000 and it was optimally active at pH 7.4 and at 50 degrees C. The Km value of the enzyme was 1.8 x 10(-3) M. Calcium, Magnesium and Zinc ions stimulated whereas Mercury ion and EDTA inhibited the enzyme activity. This enzyme exhibited fluorescence emission maximum between 310-320 nm.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Purification and characterization of a novel phospholipase A2 from king cobra (Ophiophagus hannah) venom

A novel phospholipase A₂, designated as Oh-DE-2, was isolated from the venom ofOphiophagus hannah (king cobra) by successive chromatography on SP-Sephadex C-25, DE-52, and Q-Sepharose columns. Oh-DE-2 with pI 5.1 showed an apparent molecular weight of 14 kD as revealed by SDS-PAGE and gel filtration. The amino acid sequence was homologous with those of PLA₂s from Elapidae venoms. Oh-DE-2 was effectively inactivated byp-bromophenacyl bromide, indicating that the conserved His-48 is essential for its enzymatic activity. However, modification of the conserved Trp-19 did not cause a precipitous drop in the enzymatic activity of Oh-DE-2 as observed with PLA₂s fromNaja naja atra andBungarus multicinctus venoms. A quenching study showed that the microenvironment of Trp in Oh-DE-2 was inaccessible to acrylamide, iodide, or cesium, a finding which was different from those observed with PLA₂s fromN. naja atra andB. multicinctus venoms. These results might suggest that, unlike other PLA₂ enzymes, Trp-19 in Oh-DE-2 is not directly involved in its enzymatic mechanisms.     

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated.In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.     

Purification and characterization of phospholipase A2 from rat stomach

Phospholipase A2, which is localized in the mucosal part of the corpus of rat stomach (Hirohara et al. (1987) Biochim. Biophys. Acta 919, 231-238), was purified 990-fold from the supernatant of a tissue homogenate by heat treatment at acidic pH, ammonium sulfate fractionation, ion-exchange chromatography, gel-filtration and reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The purified enzyme gave a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with a molecular mass of approx. 17 kDa. The enzyme had a pH optimum of 8.0 and hydrolyzed the 2-arachidonoyl residue of phosphatidylcholine preferentially to the 2-oleoyl residue, the Vmax and Km values for the two being 227 and 29 mumol/min per mg protein and 0.037 and 0.019 mM, respectively. The activity was calcium-dependent and was markedly increased by SDS and dimethyl sulfoxide (DMSO). The enzyme showed typical product inhibition. Free unsaturated fatty acids (oleic, arachidonic and docosahexaenoic acids), which are supposedly the main enzymatic products in vivo, inhibited the activity. Arachidonic acid caused noncompetitive inhibition and its concentration for its maximal inhibition (50% inhibition) was 5 x 10(-5) M. Lysophosphatidylcholine, free saturated fatty acids (palmitic and stearic acids) and arachidonic acid metabolites (leukotrienes and prostaglandins) had no effect on the activity.     

Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung

Molecular Biology Reports - Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin...     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

(Over)training effects on quantitative electromyography and muscle enzyme activities in standardbred horses

Too intensive training may lead to overreaching or overtraining. To study whether quantitative needle electromyography (QEMG) is more sensitive to detect training (mal)adaptation than muscle enzyme activities, 12 standardbred geldings trained for 32 wk in age-, breed-, and sex-matched fixed pairs. After a habituation and normal training (NT) phase (phases 1 and 2, 4 and 18 wk, respectively), with increasing intensity and duration and frequency of training sessions, an intensified training (IT) group (phase 3, 6 wk) and a control group (which continued training as in the last week of phase 2) were formed. Thereafter, all horses entered a reduced training phase (phase 4, 4 wk). One hour before a standardized exercise test (SET; treadmill), QEMG analysis and biochemical enzyme activity were performed in muscle or in biopsies from vastus lateralis and pectoralis descendens muscle in order to identify causes of changes in exercise performance and eventual (mal)adaptation in skeletal muscle. NT resulted in a significant adaptation of QEMG parameters, whereas in muscle biopsies hexokinase activity was significantly decreased. Compared with NT controls, IT induced a stronger adaptation (e.g., higher amplitude, shorter duration, and fewer turns) in QEMG variables resembling potentially synchronization of individual motor unit fiber action potentials. Despite a 19% decrease in performance of the SET after IT, enzyme activities of 3-hydroxyacyl dehydrogenase and citrate synthase displayed similar increases in control and IT animals. We conclude that 1) QEMG analysis is a more sensitive tool to monitor training adaptation than muscle enzyme activities but does not discriminate between overreaching and normal training adaptations at this training level and 2) the decreased performance as noted in this study after IT originates most likely from a central (brain) rather than peripheral level.     

Purification and properties of a membrane-bound phospholipase B from baker's yeast (Saccharomyces cerevisiae)

Two kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.