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PubMed2001

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.

Animals↗

PubMed1983

Effects of phospholipase A2 on gastric microsomal H+, K+-ATPase system: role of "boundary lipids" and the endogenous activator protein.

Pig gastric microsomal vesicles enriched in gastric H+,K+-ATPase and K+-pNPPase were digested with bee venom phospholipase A2 at 21 or 37 degrees C. The unattacked phospholipids were then related to the remaining enzyme activities, followed by reconstitution with microsomal phospholipids and the endogenous activator protein. Gastric K+-stimulated ATPase was nearly abolished within 10 min of phospholipase A2 treatment. A substantial amount of pNPPase activity remained unaffected under identical conditions. About 80% of the microsomal phosphatidylethanolamine was attacked by phospholipase A2 at both temperatures while 60 and 79% of the phosphatidylcholine was hydrolyzed at 21 and 37 degrees C, respectively. Analysis of the phospholipids revealed that phospholipase A2 attacked only the phosphatidylcholine and phosphatidylethanolamine molecules enriched in polyunsaturated fatty acids. Microsomal H+,K+-ATPase system inactivated by phospholipase A2 at 21 degrees C could be largely restored by the endogenous activator alone. On the other hand, those inactivated at 37 degrees C needed pretreatment with phosphatidylcholine before assaying with the activator protein for maximal reconstitution; phosphatidylethanolamine was totally ineffective in restoration of the enzyme activity. Analysis of the fatty acid composition of the lysophosphatidylcholine following phospholipase A2 treatment at 21 and 37 degrees C suggested involvement of some phosphatidylcholine molecules relatively enriched in saturated fatty acids and extremely poor in polyunsaturated fatty acids in gastric ATPase function. The data not only pointed out the importance of phosphatidylcholine and the endogenous activator in gastric microsomal H+,K+-ATPase reaction but also demonstrated considerable heterogeneity within the same species of microsomal phospholipids.

4-Nitrophenylphosphatase↗

PubMed1985

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study.

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.

Animals↗

PubMed1985

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.

Kinetics↗

PubMed1986

Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli.

A basic (pI = 10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phospholipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., & Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Evidence for the regulation of guinea-pig heart microsomal phosphatidylcholine-hydrolysing phospholipase A1 by guanosine 5'-[gamma-thio]triphosphate.

We have recently characterized lysophospholipase A2 activities in guinea-pig heart microsomes and postulated that these enzymes act sequentially with phospholipases A1 to release fatty acids selectively from phosphatidylcholine (PC) and phosphatidylethanolamine, thus providing an alternative route to the phospholipase A2 mode of release. In a further investigation of the postulated pathway, we have characterized the PC-hydrolysing phospholipase A1 in guinea-pig heart microsomes. Our results show that the enzyme may have a preference for substrates with C16:0 over C18:0 at the sn-1 position. In addition, although the enzyme cleaves the sn-1 fatty acid, the rate of hydrolysis of PC substrates with C16:0 at the sn-1 position was influenced by the nature of the fatty acid at the sn-2 position. The order of decreasing preference was C18:2 > C20:4 = C18:1 > C16:0. The hydrolyses of the molecular species were differentially affected by heating at 60 degrees C. An investigation into the effect of nucleotides on the activity of the enzyme showed that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited the hydrolysis of PC by phospholipase A1 activity, whereas GTP, guanosine 5'-[beta-thio]diphosphate (GDP[S]), GDP, ATP and adenosine 5'-[gamma-thio]triphosphate (ATP[S]) did not affect the activity. The inhibitory effect of GTP[S] on phospholipase A1 activity was blocked by preincubation with GDP[S]. A differential effect of GTP[S] on the hydrolysis of different molecular species was also observed. Taken together, the results of this study suggest the presence of more than one phospholipase A1 in the microsomes with different substrate specificities, which act sequentially with lysophospholipase A2 to release linoleic or arachidonic acid selectively from PC under resting conditions. Upon stimulation and activation of the G-protein, the release of fatty acids would be inhibited.

Animals↗

PubMed1990

Proteinaceous inhibitors of phospholipase A2 purified from inflammatory sites in rats.

We have purified two phospholipase A2 inhibitory proteins (37 and 33 kDa) from peritoneal fluid of dexamethasone-treated rats. The extracellular phospholipase A2 found in inflammatory sites differed from the exocrine phospholipase A2 in susceptibility to these endogenous inhibitors; both proteins inhibited the activity of the extracellular phospholipase A2 purified from sites of inflammation but did not affect appreciably the activity of either porcine pancreatic or Naja naja venom phospholipase A2. The amino acid sequence of the NH2-terminal portion of the purified proteins did not resemble that of lipocortins so far reported, but it was almost identical to that of parts of human or mouse complement component C3. These findings may indicate that degraded products of C3 are involved in the regulation of activity of a class of mammalian phospholipase A2.

Amino Acid Sequence↗

PubMed1996

Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2.

A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470I15470) putatively encoding a calcium-independent phospholipase A2 was expressed in S. frugiperda cells resulting in over a 15-fold increase in a calcium-independent phospholipase A1/A2 activity which was entirely inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. The recombinant polypeptide was purified from cytosol by sequential tandem affinity chromatographies employing ATP-agarose and calmodulin-Sepharose stationary phases. This strategy resulted in the rapid purification (36 h) of recombinant phospholipase A2 activity in 56% overall yield to a single intense 80-kDa protein band on SDS-polyacrylamide gel electrophoresis after silver staining. The purified protein possessed phospholipase A1, phospholipase A2, and lysophospholipase activities. Microbore anion exchange chromatography demonstrated that the 80-kDa protein band was comprised of multiple distinct isoforms including an anionic isoform which possessed over a 5-fold higher specific activity (5 micromol/mg.min) than earlier eluting isoforms. Collectively, these results unambiguously demonstrate that: 1) the 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities with distinct kinetic parameters; 2) calmodulin and ATP both interact with the catalytic polypeptide independent of regulatory proteins; and 3) distinct isoforms of this polypeptide exist which possess markedly different specific activities.

Animals↗

PubMed1990

Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom.

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.

Amino Acid Sequence↗

PubMed1984

Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists.

Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.

Acrosome↗

PubMed1986

Glucocorticoid-induced modulation of the beta-adrenergic adenylate cyclase response of epidermis: its relation to epidermal phospholipase A2 activity.

It has been suggested that glucocorticoids produce their biologic effects through the synthesis of phospholipase A2 inhibitor protein (lipocortin) in various cell systems. Recent studies from our laboratory revealed that glucocorticoids augment the beta-adrenergic adenylate cyclase response of epidermis and that this effect depends on a protein synthesis mechanism. In order to elucidate the possible mechanism of this glucocorticoid effect in terms of phospholipase A2 activity, an in vitro pig skin incubation system was employed. Mepacrine, a phospholipase A2 inhibitor, augmented the beta-adrenergic adenylate cyclase response of epidermis as glucocorticoids. The effect of mepacrine was stronger and was observed earlier than that of glucocorticoid (hydrocortisone). The addition of both mepacrine and hydrocortisone at their optimal concentrations in the incubation medium, resulted in neither an additive nor a synergistic effect on the beta-adrenergic augmentation. On the other hand, melittin, a phospholipase A2 stimulator, depressed the beta-adrenergic adenylate cyclase response. The addition of both melittin and hydrocortisone in the incubation medium resulted in the inhibition of the hydrocortisone-induced beta-adrenergic augmentation effect. Following long-term incubation with hydrocortisone, the epidermal phospholipase A2 activity was significantly decreased. These results indicate that glucocorticoids might affect the beta-adrenergic adenylate cyclase response of epidermis through the synthesis of phospholipase A2 inhibitor protein (lipocortin) as in other cell systems.

Adenylyl Cyclases↗

PubMed1980

The phospholipases A of epidermis.

Phospholipases A have been characterized in fetal rat epidermis. Both a calcium dependent phospholipase A with pH optimum of 8.5 and a calcium independent enzyme with a pH optimum of 4.5 were found. Activity against both acyl groups of phosphatidyl choline were found for both enzymes and the findings suggested that the acid enzyme (pH 4.5) is primarily phospholipase A1. The specificity of the alkaline enzyme with respect to acyl groups could not be determined due to the marked hydrolysis of lysophosphatidyl choline. Lysophospholipase activity had similar pH and calcium requirements as the alkaline phospholipase A and the 2 activities could not be dissociated in these experiments. The acid phospholipase was localized primarily in a fragile large particle fraction while the alkaline enzyme was present in various subcellular fractions and most active in the soluble supernatant. These findings demonstrate that epidermal phospholipases A are similar to those in other mammalian tissues. The presence of both acid and alkaline enzymes ar various subcellular loci suggest that the enzymes may play important roles in many facets of epidermal membrane metabolism and in particular in the degradative events of keratinization.

Animals↗

PubMed1981

Modification of arginine residues in porcine pancreatic phospholipase A2.

Although phenylglyoxal monohydrate reacts with Arg-6 in porcine pancreatic phospholipase A2, concomittantly the alpha-amino group of the N-terminal Ala-1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg-6 modification. Upon reaction of [7-(14)C]phenylglyoxal monohydrate with alpha-amino-blocked phospholipase A2 analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg-6. Removal of alpha-amino-blocking groups after the modification reaction furnished the corresponding Arg-6-modified phospholipases possessing 30-38% of their original specific enzymatic activities in the egg-yolk assay. After reaction of 1,2-[1-(14)C]cyclohexanedione with porcine phospholipase A2 the crude reaction mixture was purified by chromatography on quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of 14C-labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg-6. Cyclohexanedione modification of Arg-6 in phospholipase A2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg-6 by cyclohexanedione are in agreement with previous findings that Arg-6 is involved in the interaction of the enzyme with lipid-water interfaces.

Aldehydes↗

PubMed1989

Characterisation of soluble and cell associated phospholipase A2 from rheumatoid synovial fluid.

The hydrolysis of radiolabelled Escherichia coli phospholipids, and micellar dispersions of phosphatidylethanolamine and phosphatidylcholine, were used to characterise the phospholipase A2 activity in synovial fluid from patients with rheumatoid arthritis. Cell free fractions of synovial fluid contain a phospholipase A2 enzyme that preferentially releases [14C]oleic acid from E coli biomembranes (specific activity 291.3 (SEM 27.6) pmol/min/mg). This enzyme requires calcium and is optimally active at neutral pH. Purified dispersions of phosphatidylethanolamine are also readily degraded by the soluble enzyme, but it is not active against phosphatidylcholine. The substitution of [14C]oleic acid by [3H]arachidonic acid for the labelling of E coli allowed differentiation between the soluble phospholipase A2 and the cell associated phospholipase A2 present in sonicates of mononuclear cells and neutrophils from peripheral blood and synovial fluid. The cell associated phospholipase A2 preferentially releases [3H]arachidonic acid from E coli cardiolipin. In this paper the phospholipid substrate specificity of phospholipase A2 from rheumatoid synovial fluid, the optimal assay conditions for its detection, and a standardised expression of activity in terms of pmol per minute per mg of protein are established.

Arthritis, Rheumatoid↗

PubMed1997

Effects of phospholipase A2, 12-lipoxygenase, and cyclooxygenase inhibitors in the feline pulmonary bed.

The effects of phosphonofluoridic acid, methyl-5,8,11,14-eicosatetraenyl ester (MAFP), a phospholipase A2 inhibitor, on pressor responses to angiotensin II (ANG II), norepinephrine (NE), serotonin (5-HT), the calcium channel opener BAY K 8644, and the thromboxane A2 mimic U-46619 were studied in the pulmonary vascular bed of the intact-chest cat. Under conditions of constant lobar blood flow, injections of ANG II, NE, 5-HT, U-46619, and BAY K 8644 into the lobar arterial perfusion circuit caused dose-related increases in lobar arterial pressure that were reproducible with respect to time. Infusion of MAFP into the perfused lobar artery at doses of 100 to 300 microg/kg for 10 min significantly reduced vasoconstrictor responses to ANG II; however, the phospholipase A2 inhibitor did not alter vasoconstrictor responses to BAY K 8644, 5-HT, NE, or U-46619. The combination of the higher dose of the phospholipase A2 inhibitor MAFP with the phospholipase C inhibitor U-73122 significantly affected vasoconstrictor responses to ANG II, NE, and 5-HT but not to BAY K 8644. In a separate series of animals, the effects of a lipoxygenase inhibitor, baicalein, were investigated. Infusion of baicalein into the perfused lobar artery at doses of 100 microg/kg for 10 min significantly reduced vasoconstrictor responses to ANG II but not the vasoconstrictor responses to BAY K 8644, 5-HT, NE, or U-46619. In a final series of experiments, the effects of a cyclooxygenase inhibitor, meclofenamate, were investigated, and intravenous injection of the meclofenamate at a dose of 2.5 mg/kg did not significantly affect vasoconstrictor responses to ANG II, 5-HT, BAY K 8644, NE, or U-46619. These data provide support for the hypothesis that pulmonary vasopressor responses to ANG II are mediated, in part, by the activation of phospholipase A2, phospholipase C, and the lipoxygenase pathway in the pulmonary vascular bed of the cat.

Animals↗

PubMed1998

Hydrolysis of surfactant-associated phosphatidylcholine by mammalian secretory phospholipases A2.

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2 is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2 (sPLA2) activity is increased, but the type of sPLA2 and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2 on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (gammamin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases gammamin. In contrast, mammalian group II sPLA2 demonstrates limited hydrolysis of PC and does not increase gammamin. Group I and group II secretory phospholipases A2 from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2 from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.

Animals↗

PubMed1991

Altered phospholipase C activity in renal medulla of DOCA-salt hypertensive rats.

We investigated the alterations of phospholipase C activity in the kidney and arterial wall of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Neither DOCA nor salt administration alone affected phospholipase C activity in the kidney and arterial wall. In contrast, when the rats were given both DOCA and salt and developed high blood pressure, the phospholipase C activity became greater in the outer and inner medulla (papilla) while the enzyme activity was reduced significantly in the renal cortex and arterial wall. Such an increase in phospholipase C activity was not observed in the kidney of a genetic rat for spontaneous hypertension. Moreover, the phospholipase A2 activity was also stimulated in the renal medulla of DOCA-salt hypertensive rats. Thus, these data indicate that DOCA-salt hypertension is associated with an increase in phospholipase C activity in the renal medulla, which seems primary to the development of DOCA-salt hypertension.

Animals↗

PubMedDate not supplied

Inhibition of LDL-associated phospholipase A activity in human plasma by albumin.

LDL has been previously shown to exhibit activity of phospholipase A1 and phospholipase A2 as measured by the hydrolysis of NBD caproic phosphatidylcholine (C6-NBD-PC), which is considered to represent the phospholipase activity towards short chain or oxidized fatty acyl chains/3/. In the present study we show that in whole plasma the LDL-associated phospholipase A activity, measured by the hydrolysis of C6-NBD-PC, is inhibited by albumin due to its binding of the substrate. The inhibition depends on the molar ratio between the substrate, albumin and the enzyme. C6-NBD-PC and other synthetic phospholipid analogues have been used previously to determine plasma phospholipase A2 activity in various pathological states. This study suggests that when using this kind of substrate to measure plasma PLA activity, the choice of substrate and the experimental conditions should be carefully considered. Determination of the appropriate ratio between the three reaction components--enzyme, substrate and albumin--is required for reliable and consistent determination of plasma phospholipase activity.

Humans↗