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PubMed1993

Hepatic insulin resistance in KKA(y) mice and its amelioration by pioglitazone do not involve alterations in phospholipase C activity.

It has been proposed that an abnormality in the regulation of cytosolic-free Ca2+ may be the cause of some forms of insulin resistance. In support of this proposition, it was reported that phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by liver plasma membranes from obese patients with non-insulin-dependent diabetes mellitus (NIDDM) was abnormally augmented (Thakker et al., J. Biol. Chem. 264, 7169-7175). The objective of this investigation was to determine if a novel antidiabetic agent, pioglitazone, ameliorated hepatic insulin resistance in KKA(y) mice and to identify any alterations in PIP2-phospholipase C activity of liver plasma membranes that may accompany changes in insulin sensitivity. Treatment of KKA(y) mice for 4 days with pioglitazone (20 mg/kg per day) decreased blood glucose and insulin and improved a variety of indices of hepatic insulin resistance, but did not alter the rate of PIP2 hydrolysis by liver plasma membranes. Acute treatment of isolated liver plasma membranes with pioglitazone (1-100 microM) also failed to alter PIP2-phospholipase C activity. Furthermore, the specific activity, Ca(2+)-requirement, pH-dependence and sensitivity to guanosine 5'-thiotriphosphate of the PIP2-phospholipase C in KKA(y) liver membranes were indistinguishable from those of C57BL/6J (normal) mice. Among C57BL/6J and KKA(y) mice fed either a control or pioglitazone-supplemented diet, there was no correlation between PIP2-phospholipase C activity in isolated liver membranes and either glucose or insulin concentrations in the circulation. These data indicate that an alteration in PIP2-phospholipase C activity of liver plasma membranes is neither a cause nor an obligatory consequence of insulin resistance in KKAy mice or its amelioration by pioglitazone. Alterations of liver membrane phospholipase C activity in NIDDM, therefore, may reflect diabetic pathology other than the insulin resistance associated with this disease.

Animals↗

PubMed1996

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

Animals↗

PubMed2005

Sol i 1, the phospholipase allergen of imported fire ant venom.

BACKGROUND: Sol i 1, the venom phospholipase of imported fire ant venom is an important allergen and exhibits some cross-reactivity with IgE antibodies from patients sensitized to other Hymenoptera venoms. OBJECTIVE: To determine the primary structure of Sol i 1 and evaluate the roles of protein and carbohydrate epitopes in its cross-reactivity. METHODS: Sol i 1 was purified from venom, proteolytic peptides prepared and amino acid sequences obtained. The cDNA for Sol i 1 was cloned, sequenced, and compared with sequences of other wasp venom phospholipases. The role of carbohydrate epitopes in the cross-reactivity with other Hymenoptera venoms was studied by RAST inhibition. RESULTS: The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. Mannosylated N-glycans could completely inhibit binding of IgE from honeybee venom sensitized patients to Sol i 1. Inhibition by glycan of IgE binding from yellow jacket venom sensitized patients was low or absent for three of eight sera and substantial, but not complete for five sera. CONCLUSIONS: Sol i 1 is related to wasp venom phospholipases. Cross-reactivity with honeybee venom is caused by carbohydrate, whereas cross-reactivity with yellow jacket venom involves reactivity with both carbohydrate determinants of hyaluronidase and high molecular weight proteins and phospholipase protein determinants.

Allergens↗

PubMed1991

Phospholipase C activity in microorganisms associated with reproductive tract infection.

Phospholipase C (lecithinase or phosphatidylcholine phosphorylase) catalyzes the hydrolysis of lecithin into phosphorylcholine and 1,2-diglyceride. Bacterial production of phospholipase C may damage reproductive tract tissues by both direct and indirect mechanisms. Use of the synthetic substrate p-nitrophenylphosphorylcholine phospholipase C activity was determined in 204 isolates representative of those found in female genital tract. Multiple aerobic (28%) and anaerobic (28%) reproductive tract microorganisms showed phospholipase C activity. Phospholipase C-producing isolates included strains of Bacteroides fragilis, B. bivius, B. thetaiotaomicron, Gardnerella vaginalis, and group B streptococcus. Phospholipase C activity was heterogenous; not all isolates that belong to a particular species showed activity. Phospholipase C production may be a possible virulence factor produced by a number of microflora commonly implicated in various reproductive tract infections or conditions, as well as in some instances of preterm birth.

Bacteria, Aerobic↗

PubMed2003

A continuous spectrophotometric assay for phospholipase A(2) activity.

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).

Deoxycholic Acid↗

PubMed2001

Effects of phospholipase A(2) activating peptides upon GTP-binding protein-evoked adrenocorticotrophin secretion.

A GTP-binding protein (G-protein), termed G-exocytosis (Ge), mediates the effects of calcium ions in the late stages of the adrenocorticotrophin (ACTH) secretory pathway. An activator of Ge, mastoparan, also stimulates phospholipase A(2) and so a comparison of other phospholipase A(2)-activating peptides, melittin and phospholipase A(2)-activating peptide was made with mastoparan to assess whether phospholipase A(2)activation was an important component of Ge-evoked secretion. All three peptides stimulated ACTH secretion in the effective absence of calcium ions from permeabilised cells, actions potentiated by a phospholipase A(2)inhibitor. Ca(2+)-evoked secretion from permeabilised cells was similarly potentiated by a phospholipase A(2) inhibitor. Furthermore, arachidonic acid inhibited Ca(2+)- and Ge-evoked ACTH secretion, an action blocked by the cyclo-oxygenase inhibitor ibuprofen. This study suggests that the products of phospholipase A(2)-generated arachidonic metabolism may exert an inhibitory action on the late post-Ca(2+) stages of the ACTH secretory pathway and that prostaglandins may be the active agents in this capacity.

Adrenocorticotropic Hormone↗

PubMed2001

Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation.

Muscarinic receptor agonists transiently activate phospholipase D in tracheal smooth muscle. Muscarinic activation of phospholipase D in this tissue is dependent on activation of protein kinase C and an unidentified pathway that is not protein kinase C dependent. Cholinergic agents have also been shown to activate phospholipase D by pathways linked to the small G protein, RhoA. This study explores the relationship between muscarinic activation of phophatidylinositol 3-kinase and activation of RhoA, and examines whether phospholipase D activation is dependent on either pathway in tracheal smooth muscle. Wortmannin or 2-(4-morphonyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), putative specific inhibitors of phophatidylinositol 3-kinase, significantly inhibit acetylcholine-induced formation of phosphatidylethanol and also block acetylcholine-induced translocation of RhoA to the membrane. In previous experiments calphostin C, a protein kinase C inhibitor, partially inhibited both acetylcholine-induced and phorbol-12-myristate-13-acetate (PMA)-induced phosphatidylethanol formation. In the present study calphostin C did not block acetylcholine-induced RhoA translocation to the membrane. However, the Rho kinase inhibitor, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), significantly inhibited acetylcholine-induced phosphatidylethanol formation, but had no effect on activation of phospholipase D by PMA. Acetylcholine treatment also stimulated the phosphorylation of the 110-kDa subunit of phosphatidylinositol 3-kinase. Phosphorylation of phosphatidylinositol 3-kinase 110-kDa subunit could be blocked by wortmannin in a concentration-dependent manner, and acetylcholine-induced phosphatidylinositol 3-kinase activity was significantly inhibited by wortmannin. LY-294002 also inhibited acetylcholine-induced phosphorylation of 110-kDa subunit and activation of phosphatidylinositol 3-kinase. These results suggest that acetylcholine stimulation translocates RhoA to the membrane by a phosphatidylinositol 3-kinase-dependent mechanism and acetylcholine-induced phospholipase D stimulation is at least partly mediated via phosphatidylinositol 3-kinase, however, protein kinase C appears to activate phospholipase D independent of phosphatidylinositol 3-kinase or RhoA activation in porcine tracheal smooth muscle.

Acetylcholine↗

PubMed2000

Transcriptional regulation of inflammatory secreted phospholipases A(2).

Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene.

Animals↗

PubMed2003

Activation of phospholipase D is not mediated by direct phosphorylation on tyrosine residues.

The activation of phospholipase D (PLD) in PC12/PC2 pheochromocytoma cells involves a tyrosine kinase. However, it is not clear whether this is due to direct phosphorylation of the enzyme or some other intermediary protein. In this manuscript, we examined this issue by two methods: (1) immunoprecipitation of phosphotyrosine containing proteins and assay of phospholipase D; (2) overexpression of HA-phospholipase D2 and susbsequent immunoprecipitation. The only agent that caused phosphorylation of phospholipase D on tyrosine residues was the phosphatase inhibitor, peroxyvanadate. Other agents that activate phospholipase D, including bradykinin, ionomycin, and phorbol dibutyrate did not cause phosphorylation of the enzyme. In addition, there was a lack of correlation between the peroxyvanadate-mediated phosphorylation and activation of phospholipase D, both in terms of time course and concentration dependence. These data demonstrate that phospholipase D is directly phosphorylated on tyrosine residues. However, phosphorylation of tyrosine residues does not correlate with activation of the enzyme.

Animals↗

PubMed1999

Effects of phosphatidylinositol 4,5-bisphosphate and neomycin on phospholipase D: kinetic studies.

The kinetics of phosphatidylcholine-specific phospholipase D activated by phosphatidylinositol 4,5-bisphosphate (PIP2) and inhibition by neomycin were studied in an enzyme preparation partially purified from human hepatocarcinoma cell line. It was found that phospholipase D was marginally activated by phosphatidyl-4-phosphate (PIP) and phosphatidylethanolamine (PE). In contrast, it was considerably activated by PIP2 in different concentration of phosphatidylcholine (PC). Sphingomyelin (SM), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were neither substrates nor inhibitors of the phospholipase D. PIP, induced an allosteric effect on phospholipase D and a negative cooperative effect with respect to phosphatidylcholine as indicated in the Lineweaver-Burk plot. In the absence of PIP2, a straight line was obtained, whereas a downward concave curve was observed in the presence of 25 microM of PIP2. The Hill coefficient and the apparent K(m) of phosphatidylcholine in the presence of 25 microM PIP, were calculated to be 0.631 and 10.79 mM, respectively. PIP2 also increased the maximal velocity (Vmax) of the phospholipase D reaction, suggesting that the affinity of substrate to enzyme was decreased, and the turnover number of the enzyme (kcat) was increased by PIP2. The activation of phospholipase D by PIP2 was dose dependent up to 50 microM of PIP2. The Ka of PIP2 was 15.8 mM. Neomycin, a polycationic glycoside, was shown to be an uncompetitive inhibitor of phospholipase D, and revealed the formation of a neomycin-PIP2 complex. The Ki of neomycin was estimated to be 8.7 mM.

Carcinoma, Hepatocellular↗

PubMed1986

Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets.

The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.

Blood Platelets↗

PubMed1993

The substrate specificity of brain microsomal phospholipase D.

Neurotransmitters activate a phospholipase D that is though to specifically hydrolyse phosphatidylcholine. This enzyme has a unique property known as transphosphatidylation: in the presence of an appropriate nucleophilic receptor such as an alcohol, phospholipase D will catalyse the production of phosphatidyl-alcohol. We have studied phospholipase D using an in vitro assay that uses [3H]butanol of high specific radioactivity (15 Ci/mmol) as an acceptor. In the presence of [3H]butanol and phosphatidylcholine, a microsomal membrane fraction from rat brain catalysed the production of phosphatidyl[3H]butanol. Phospholipase D activity was dependent upon the presence of a detergent; the optimal sodium oleate concentration was between 4 and 6 mM. The RF of the phosphatidyl[3H]butanol on t.l.c. was identical to the RF of the phosphatidylbutanol formed when [3H]phosphatidylcholine was incubated with 100 mM butanol. These data confirm the identity of phosphatidyl[3H]butanol. One important advantage of this assay is that the substrate does not need to be labelled. We have used this advantage to examine the substrate specificity of phospholipase D. Microsomal phospholipase D appears to hydrolyse phosphatidylcholine most efficiently. There is a relatively small but significant activity against phosphatidylethanolamine and phosphatidylserine, and there is no significant activity against phosphatidylinositol. As the head-group becomes more like choline, the phospholipid becomes a better substrate for phospholipase D. The addition of one methyl group leads to a large increase in activity. Fatty acid composition does not play a role in determining the substrate specificity. This assay should be useful in furthering our understanding of this important enzyme.

Animals↗

PubMed1994

Suppression of Gi alpha 2 enhances phospholipase C signalling.

G-proteins mediate transmembrane signalling from a populous group of cell-surface receptors to a smaller group of effectors that includes adenylate cyclase, various ion channels and phospholipase C. Stem cells (F9 teratocarcinoma) or rat osteosarcoma 17/2.8 cells in which Gi alpha 2 expression is abolished by antisense RNA display markedly elevated basal inositol 1,4,5-trisphosphate accumulation and a potentiated phospholipase C response to stimulatory hormones. Expression of the Q205L mutant of Gi alpha 2, which is constitutively active, was found to block persistently hormonally stimulated phospholipase C activity, implicating Gi alpha 2 as an inhibitory regulator of phospholipase C signalling. Analysis using Gi alpha 2-deficient adipocytes of transgenic mice provided further evidence for a role for Gi alpha 2 in phospholipase C regulation, demonstrating in vivo that loss of Gi alpha 2 elevates basal, and markedly potentiates hormonally stimulated, phospholipase C activity. This report demonstrates for the first time that a single G-protein, G12, can regulate two distinct signalling pathways, i.e. adenylate cyclase and phospholipase C.

Adipocytes↗

PubMed1994

Inositol-specific phospholipase D activity in health and disease.

1. We report the first demonstration of the pathophysiological importance and clinical applications of the relatively recently discovered circulating enzyme, phosphoinositol-specific phospholipase D. This enzyme is known to cleave the large variety of important cell-surface molecules linked to the cell membrane by glycan-phosphatidylinositol linkages (glycan-phosphatidylinositol anchors). 2. When measured in the sera of healthy individuals, phosphoinositol-specific phospholipase D activity was found to show a strong negative correlation with age, the degree of depreciation being greater than that measured for most other analytes. 3. Serum phosphoinositol-specific phospholipase D activity was considerably depressed in patients presenting with conditions leading to reduced liver synthetic reserve, such as hepatocellular carcinoma or liver cirrhosis caused by chronic viral hepatitis, and correlated with reduced albumin levels in these conditions, indicating that the liver is the site of phosphoinositol-specific phospholipase D synthesis and that phosphoinositol-specific phospholipase D may be used as an additional marker of liver synthetic reserve. 4. When measured in patients with acute liver disease, such as acute viral hepatitis, or in patients with bronchopneumonia, phosphoinositol-specific phospholipase D activity was found to be significantly raised, demonstrating features characteristic of an acute-phase reactant. 5. These findings indicate that, besides its pathophysiological importance, phosphoinositol-specific phospholipase D and the measurement of its activity in serum may have a useful place in the investigation of a range of clinical conditions, including tissue injury and inflammation.

Acute Disease↗

PubMed1995

Histamine H1 and endothelin ETB receptors mediate phospholipase D stimulation in rat brain hippocampal slices.

Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans-(1S,3R)-aminocyclopentyl-1,3-dicarboxylic acid (trans-ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans-ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 microM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 nM) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

Animals↗

PubMed2004

A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis.

During phosphate starvation, it is known that phospholipids are degraded, and conversely, a nonphosphorus galactolipid digalactosyldiacylglycerol accumulates in the root plasma membrane of plants. We report a novel phospholipase C that hydrolyzes phosphatidylcholine and is greatly induced in response to phosphate deprivation in Arabidopsis. Since phosphatidylcholine-hydrolyzing activity by phospholipase C was highly up-regulated in phosphate-deprived plants, gene expression of some phospholipase C was expected to be induced during phosphate starvation. Based on amino acid sequence similarity to a bacterial phosphatidylcholine-hydrolyzing phospholipase C, six putative phospholipase Cs were identified in the Arabidopsis genome, one of which, NPC4, showed significant transcriptional activation upon phosphate limitation. Molecular cloning and functional expression of NPC4 confirmed that the NPC4 gene encoded a functional phosphatidylcholine-hydrolyzing phospholipase C that did not require Ca(2+) for its activity. Subcellular localization analysis showed that NPC4 protein was highly enriched in the plasma membrane. Analyses of transferred DNA-tagged npc4 mutants revealed that disruption of NPC4 severely reduces the phosphatidylcholine-hydrolyzing phospholipase C activity in response to phosphate starvation. These results suggest that NPC4 plays an important role in the supply of both inorganic phosphate and diacylglycerol from membrane-localized phospholipids that would be used for phosphate supplementation and the replacement of polar lipids in the root plasma membrane during phosphate deprivation.

Amino Acid Sequence↗

PubMed2004

Comparison of extracellular phospholipase activities in clinical and environmental Aspergillus fumigatus isolates.

Extracellular phospholipase production by environmental and clinical isolates of Aspergillus fumigatus collected from several centres world-wide were compared. All isolates produced extracellular phospholipases which included phospholipase C and a phospholipid acyl hydrolase (phospholipase A and/or phospholipase B) activity. Clinical isolates of A. fumigatus produced the largest zone sizes in a diffusion assay and clinical isolates produced more extracellular phospholipase C than environmental isolates. However, environmental isolates of A. fumigatus showed increased acyl hydrolase activity compared to clinical isolates of A. fumigatus. This study suggests that extracellular phospholipase C activity, but not extracellular acyl hydrolase activity may be important in the pathogenicity of A. fumigatus.

Aspergillus fumigatus↗

PubMed1973

Lysosomal phospholipase A 1 and A 2 of normal and bacillus calmette guerin-induced alveolar macrophages.

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5-4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, beta-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A(1) and A(2) which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50-70% by 2.0 mM CaCl(2). Homogenates of BCG-induced macrophages had specific activities of beta-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 10(7) cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

Acid Phosphatase↗