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Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP 3 . In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation of many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions. In this review, we discuss current knowledge of the principle types of PPIn-protein interactions, focusing on specific lipid-binding domains. We then discuss how these domains have been re-tasked by biologists as molecular probes for these lipids in living cells. Finally, we describe how the knowledge gained with these probes, when combined with other techniques, has led to the current view of the lipids' localization and function in eukaryotes, focusing mainly on animal cells. This article is part of a Special Issue entitled Phosphoinositides., (Published by Elsevier B.V.)

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly "from blood to gut" and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen., (Copyright © 2011 Elsevier B.V. All rights reserved.)

α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.

PlcHR(2) is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR(2) when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR(2) cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR(2) on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR(2)-induced liposome aggregation, as long as Ca(2+) is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.

Clostridium perfringens alpha-toxin (370 residues) is a major virulence factor in the pathogenesis of gas gangrene. The toxin is composed of an N-terminal domain (1-250 residues) where lies the catalytic site and a C-terminal domain (251-370 residues), the Ca(2+)-binding domain, responsible for binding to membranes. The role of Tyr-57 and Tyr-65 close to the catalytic pocket (site) in the N-domain was investigated. Replacement of Tyr-57 and -65 with alanine, leucine, or phenylalanine did not affect the sphingomyelinase activity of the toxin for sodium deoxycholate-solubilized shingomyelin. However, the substitution of Tyr-57 and -65 with alanine or leucine resulted in a radical reduction in the hemolysis of sheep erythrocytes, the release of carboxyfluorescein from shingomyelin-cholesterol (1:1) liposomes, and a significant decrease in binding to the liposomes. The binding of variant toxins, Y57C/C169L and Y65C/C169L, labeled with the environmentally sensitive fluorophore, acrylodan, to the liposomes suggested insertion of the variants in a hydrophobic environment in the bilayer. These observations suggested that Tyr-57 and -65 play a role in the penetration of the toxin into the bilayer of membranes and access of the catalytic site to sphingomyelin in membranes, but do not participate in the enzymatic activity.

When human platelets are chilled below 20 degrees C, they undergo cold-induced activation. We have previously shown that cold activation correlates with the main phospholipid phase transition (10-20 degrees C) and induces the formation of large raft aggregates. In addition, we found that the glycoprotein CD36 is selectively enriched within detergent-resistant membranes (DRMs) of cold-activated platelets and is extremely sensitive to treatment with methyl-beta-cyclodextrin (MbetaCD). Here, we further studied the partitioning of downstream signaling molecules within the DRMs. We found that the phospholipase Cgamma2 (PLCgamma2) and the protein tyrosine kinase Syk do not partition exclusively within the DRMs, but their distribution is perturbed by cholesterol extraction. In addition, PLCgamma2 activity increases in cold-activated cells compared to resting platelets and is entirely inhibited after treatment with MbetaCD. The Src-family protein tyrosine kinases Src and Lyn preferentially partition within the DRMs and are profoundly affected by removal of cholesterol. These kinases are non-redundant in cold-activation. CD36, active Lyn, along with inactive Src and PLCgamma2 co-localize in small raft complexes in resting platelets. Cold-activation induces raft aggregation, resulting in changes in the activity of these proteins. These data suggest a crucial role of raft aggregation in the early events of cold-induced platelet activation.

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C beta1 (PLC beta1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC beta1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5', showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.

Using endogenous lipid substrates, assays of lipid phosphorylation indicated that neuronal nuclei had a considerable superiority in phosphatidic acid (PA) formation when compared with homogenates and other subfractions of cerebral cortex. This predominance of neuronal nuclear PA labelling was linked to a sizable pool of nuclear diacylglycerols that expanded significantly with incubation. PA was also the dominant product of neuronal nuclear lipid phosphorylation reactions. Nuclear envelope preparations and the parent neuronal nuclei showed specific rates of PA formation that were comparable, based upon membrane phospholipid contents. As well, using an exogenous diacylglycerol substrate, the distribution of diacylglycerol kinase activities closely followed phospholipid contents of subfractions derived from the neuronal nucleus during envelope preparation. This evidence suggested an association between diacylglycerol kinase and the neuronal nuclear envelope. Nuclear PA formation increased in the presence of sphingosine, while sphingosine decreased PA formation in other subfractions. Likely sphingosine exerted its effect on nuclear diacylglycerol kinase, as sphingosine did not elevate levels of nuclear diacylglycerols. Phosphoinositidase C was present in the nuclei and inhibitors of this enzyme did decrease PA formation, indicating diacylglycerols from inositides as substrates for nuclear diacylglycerol kinase. The nuclear envelope fraction had a considerably lower specific phosphoinositidase C activity than the parent nuclei, and showed an activation of PA formation by sphingosine, but a less efficient handling of the exogenous diacylglycerol substrate. It is possible that phosphoinositidase C and diacylglycerol kinase are closely situated within the neuronal nuclei, and a loss of the former activity may compromise the latter.

Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

The phosphatidylcholine (PC)-preferring phospholipase C (PLC) from Bacillus cereus (PLC(Bc)) hydrolyzes various 1,2-diacyl derivatives of PC at different rates. Substrates with side chains having eight or more carbons are present in micellular form in aqueous media and are processed most rapidly. The catalytic efficiency (k(cat)/K(m)) for the hydrolyses of short-chain PCs at concentrations below their respective critical micelle concentrations also decreases as the side chains become shorter, and this loss of efficiency owes its origin to increases in K(m). In order to ascertain whether the observed increases in K(m) might arise from conformational changes in the glycerol backbone, nuclear magnetic resonance (NMR) experiments were performed in D(2)O to determine the (3)J(HH) and (3)J(CH) coupling constants along the glycerol subunit of 1, 2-dipropanoyl-sn-glycero-3-phosphocholine (K(m)=61 mM), 1, 2-dibutanoyl-sn-glycero-3-phosphocholine (K(m)=21.2 mM) and 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (K(m)=2.4 mM). Using these coupling constants, the fractional populations for each rotamer about the backbone of each of substrate were calculated. Two rotamers, which were approximately equally populated, about the sn-1-sn-2 bond of each substrate were significantly preferred, and in these conformers, the oxygens on the sn-1 and sn-2 carbons of the backbone were synclinal to optimize intramolecular hydrophobic interactions between the acyl side chains. There was greater flexibility about the sn-2-sn-3 bond, and each of the three possible staggered conformations was significantly populated, although there was a slight preference for the rotamer in which the oxygen bearing the phosphate head group was synclinal to the oxygen at the sn-2 carbon and to the sn-1 carbon; in this orientation, the head group is folded back relative to the side chains. These studies demonstrate that there is no significant change in the conformation about the glycerol backbone as a function of side chain length in short-chain phospholipids. Thus, prior organization of the substrate seems an unlikely determinant of the catalytic efficiency of PLC(Bc), and other factors such as hydrophobic interactions or differential solvation/desolvation effects associated with the complexation of the substrate with PLC(Bc) may be involved.

Mammalian phosphoinositide-specific phospholipases C (PI-PLCs) are involved in most receptor-mediated signal transduction pathways. The mammalian isozymes employ a modular arrangement of domains to achieve a regulated production of two key second messengers. The roles of the PH, EF hand, C2, SH2 and SH3 modules in regulation of these enzymes and in interactions with membranes and other proteins is becoming apparent from recent structural and functional studies.

The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

We report a unique isoform of PLCbeta4 in rat, PLCbeta4c, that has an additional 37-nucleotide exon inserted between nucleotides 3459-3460 of the previously published PLCbeta4a coding sequence. This insertion results in replacement of 22 amino acid residues at the carboxyl terminal tail of PLCbeta4a with 41 unique residues. A human EST for PLCbeta4 also contains this exon and this exon was mapped to within a 5.5 kb intron of the human PLCbeta4 gene. PLCbeta4c is the third PLCbeta4 isoform to be identified which has a unique carboxyl-terminal tail. PLCbeta4b differs from PLCbeta4a by truncation 162 amino acid residues from the carboxyl terminus which are replaced with 10 distinct amino acid residues. Reverse transcription-polymerase chain reaction experiments show that both PLCbeta4a and PLCbeta4c mRNA are expressed throughout the rat brain and that PLCbeta4c mRNA is highly expressed in the eye and cerebellum. RNase protection assays demonstrate that both PLCbeta4a and PLCbeta4c transcripts are abundant in the cerebellum. The different carboxyl terminal tails of PLCbeta4 isoforms may allow for differential targeting and subcellular localization, contributing to regulation of PLC beta4-mediated signal transduction.

A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

A cDNA encoding mouse PLC-beta3 (mPLC-beta3) was identified by screening a mouse kidney cDNA library and using the rapid amplification of cDNA ends (RACE) method. The predicted open reading frame was 3705 bp in length. The deduced 1235 amino acid (aa) sequence shares 95.3% and 92% homology with the sequences of rat and human PLC-beta3, respectively. The corresponding mRNA is highly expressed in kidney, skeletal muscle, liver, lung, heart and brain. In spleen, mPLC-beta3 mRNA was not detectable, which is in contrast to humans where there is a distinct expression. Using ultrastructural immunocytochemistry, mPLC-beta3 expression was detected in the heterochromatin of the nucleus in mouse brain neurons. The observation of PLC-beta3 nuclear localization suggests that PLC-beta3 may have intranuclear functions.

The regulation of Phospholipase C (PLC)delta activity remains obscure. These studies show that PLCdelta1 activity is significantly enhanced by both guanosine thiotriphosphate (GTPgammaS) and Clostridium botulinum exoenzyme C3 (C3) but not by aluminium fluoride. C3 ADP ribosylated a 21-kDa protein in the PLCdelta1 preparation and Western blotting identified rhoA in these samples. RhoA acts as an inhibitory modulator of PLCdelta activity., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes., (Copyright 1998 Elsevier Science B.V.)

Phospholipase C-beta1 (PLC-beta1), one of the PLC-beta isozymes, exists as two immunologically distinguishable polypeptides of 150 (PLC-beta1a) and 140 kDa (PLC-beta1b) which are encoded in two distinct transcripts and generated by alternative splicing of a single gene. In this study, the subcellular localization of the two phospholipases C-beta1 proteins was examined in rat C6Bu-1 glioma cells using immunological techniques. Immunoblot analysis revealed that the two forms of PLC-beta1 were detectable in both cytosolic and nuclear fractions. PLC-beta1a appeared to be located preferentially in the cytosol, whereas PLC-beta1b was found predominantly in the nuclei of C6Bu-1 cells. Immunocytochemical experiments confirmed the differential localization of the two PLC-beta1 species in C6Bu-1 cells. These results suggest that the two PLC-beta1 proteins may have different physiological roles in the cell.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

A number of enzymes are known to release alkaline phosphatase and other glycan phosphatidylinositol-anchored proteins from membrane surfaces. We describe a novel approach to detect and measure these activities by partitioning in aqueous phase systems. The procedures avoid the complications of micelle-formation involving hydrophobic molecules that may arise with detergent-based partition systems and can clearly distinguish between inositol-specific phospholipase C and D activities.

The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration. For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position. The length of the ester or ether chains is of minor importance. Any deviation from these structural requirements results in a large decrease in the hydrolysis rate. These essential structural and configurational elements for optimal activity for the B. cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.

By use of carboxyfluorescein-loaded multilamellar liposomes prepared from synthetic phosphatidylcholine (PC) or sphingomyelin and cholesterol in a molar ratio of 1:1, we studied whether or not fatty acyl domain of the phospholipids affects the membrane-damaging action (or channel formation) of Staphylococcus aureus alpha-toxin on the phospholipid-cholesterol membranes. Our data indicated: (1) that toxin-induced carboxyfluorescein-leakage from the liposomes composed of saturated fatty acyl residue-carrying PC and cholesterol was decreased with increasing chain length of the acyl residues between 12 and 18 carbon atoms, although toxin-binding to the liposomes was not significantly affected by the length of fatty acyl residue; (2) that unsaturated fatty acyl residue in PC or sphingomyelin molecule conferred higher sensitivity to alpha-toxin on the phospholipid-cholesterol liposomes, compared with saturated fatty acyl residues; and (3) that hexamerization of alpha-toxin, estimated by SDS-polyacrylamide gel electrophoresis, occurred more efficiently on the liposomes composed of PC with shorter fatty acyl chain or unsaturated fatty acyl chain. Thus, hydrophobic domain of the phospholipids influences membrane-channel formation of alpha-toxin in the phospholipid-cholesterol membrane, perhaps by modulating packing of phospholipid, cholesterol and the toxin in membrane.

The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.