Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C.

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.     

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.     

Cholinesterase solubilizing factor from Cytophaga sp. is a phosphatidylinositol-specific phospholipase C

In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles.     

Ectoenzyme release from rat liver and kidney by phosphatidylinositol-specific phospholipase C

Ectoenzyme release from rat liver and kidney by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphatase and 5'-nucleotidase were released from rat kidney slices to extents of up to 60% and 30%, respectively. Release of alkaline phosphatase was observed at lower amounts of PI-specific phospholipase C than that of 5'-nucleotidase. Both enzymes were more easily released from microsomal fractions or free cells. From kidney cells, alkaline phosphatase was released without cell lysis, and more than 80% release of alkaline phosphatase was observed at 3.8% hydrolysis of PI. Isoelectric focusing profiles of alkaline phosphatase released by PI-specific phospholipase C were significantly different from the control in the cases of both rat liver and kidney. Lubrol-solubilized alkaline phosphatase was eluted at the void volume of a Toyopearl HW-55 column, while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the lower-molecular-weight region corresponding to 100,000-110,000 daltons. Furthermore, Lubrol-solubilized phosphatase became more thermostable on treatment with PI-specific phospholipase C.     

NAD glycohydrolase: enzyme characterization using intact mammalian erythrocytes

1. NADase activity has been determined on intact erythrocytes of several species. 2. Although a wide range in maximum velocity exists across species, Michaelis constants observed are very similar. 3. The enzyme is found on the outer surface of the erythrocyte plasma membrane. 4. It is inhibited by substrate after an apparent permanent modification. 5. This modification may be due to self ADP-ribosylation. 6. We have also demonstrated the presence of an ADP-ribosyltransferase on the outer surface of the sheep erythrocyte membrane.     

Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.     

Membrane-associated phospholipase C of Drosophila retina

Phospholipase C activities against phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol have been examined using head homogenate of Drosophila visual mutants. In many mutants the enzyme activities were found to be reduced. The activities against both phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol were always affected in parallel among the mutants, while the activities of other enzymes related to phosphatidylinositol metabolism, such as diacylglycerol kinase, were not. The enzyme was concluded to be membrane-associated and was activated maximally at low Ca2+ concentration (10(-7) M), when phosphatidylinositol 4,5-bisphosphate was used as a substrate, while the activity obtained with phosphatidylinositol increased with the Ca2+ concentration up to 10(-4) M. The effects of pH on these two enzyme activities differed to some extent.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Axolotl pronephric duct cell migration is sensitive to phosphatidylinositol-specific phospholipase C

On the basis of its distribution pattern in embryos of the axolotl (Ambystoma mexicanum), we recently identified alkaline phosphatase as a molecule potentially involved in guiding the migration of the pronephric duct. Alkaline phosphatase is a cell surface protein anchored to cell membranes via a covalent linkage to a phosphatidylinositol glycan (PI-G). The enzyme phosphatidylinositol-specific phospholipase C (PIPLC) specifically releases from cell surfaces molecules anchored by the PI-G linkage. In order to test the possibility that a PI-G anchored protein is involved in directing pronephric duct cell migration, PIPLC was applied to axolotl embryos. The enzyme was introduced into embryos through the use of a novel slow-release bead material, hydrolysed polyacrylamide. PIPLC blocked pronephric duct cell migration without interfering with somite fissure formation, a concurrent, neighbouring morphogenetic cell rearrangement which occurs with little if any alkaline phosphatase present. In addition, alkaline phosphatase activity was markedly diminished in the vicinity of the implanted beads. These observations suggest that at least one protein anchored to the cell membrane by a PI-G linkage, possibly alkaline phosphatase, is involved in guiding or promoting pronephric duct cell migration.     

Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C.

Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.     

Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Sirtuins are NAD (+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD (+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces alpha-stereochemistry as established by reaction of methanol which forms alpha-1- O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate beta-stereochemistry. Stereochemistry was established by isolation of beta-1- O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.     

Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

4,4-Di-isothiocyanostilbene-2,2'-disulphonic acid inhibition of taurocholate efflux from canalicular vesicles was used to demonstrate that potential driven and 'carrier'-mediated canalicular excretion of taurocholate occur via a common, rather than two separate, pathways. This electrogenic canalicular bile acid 'carrier' preferentially transports trihydroxylated and conjugated dihydroxylated bile acids, but not the unphysiological oxo bile acids, and possibly extends its substrate specificity to other amphipathic molecules such as sulphobromophthalein.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine platelets

Phosphatidylinositol-specific phospholipase C was purified to homogeneity from soluble fraction of bovine platelets by ammonium sulfate fractionation, hydrophobic chromatography, DEAE ion exchange chromatography and gel filtration. The purified enzyme has a narrow pH optimum ranging from 6.5 to 7.5 and the molecular weight of the enzyme was estimated to be 143,000 by sodium dodecyl sulfate slab gel electrophoresis. The purified enzyme requires Ca2+ strictly for activity, which was markedly enhanced in the presence of arachidonate. No enhancement of the activity was observed in the presence of purified calmodulin. The activity was markedly inhibited in the presence of quinacrine but no inhibition by indomethacin was observed.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.     

A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.     

Activation of calcium transport in synaptosomes by phosphatidylinositol-specific phospholipase C

The isotope labeling method was used to study the influence of phospholipases C of different origin and specificity on Ca2+ accumulation in rat brain synaptosomes. It was found that phospholipases C specific to phosphatidylinositides (PI) stimulate Ca2+ transport into synaptosomes, while non-specific phospholipase C, which hydrolyzes different membrane lipid fractions, decreases the Ca2+ content in synaptosomes. It is supposed that the stimulating effect of PI-specific phospholipases C is determined by the activation of PI metabolism, which results in an increase in the content of some PI metabolism products serving as Ca2+ ionophores in synaptosomal membranes. The inhibition of Ca2+ uptake by synaptosomes treated with non-specific phospholipase C is thought to result from partial disruption of synaptosomal membranes.     

Immunohistochemical localization of NAD glycohydrolase in human and rabbit tissues

NAD glycohydrolase (NADase) is present in many organisms from bacteria to mammals. In any given organism, this enzyme is ubiquitous in many tissues. However, its precise localization and its physiological significance have not been defined. We have determined the distribution of NADase in normal human and rabbit tissues by immunoblotting and immunohistochemistry, using a polyclonal antibody raised in goats. Immunoblot analyses revealed that NADase was highly expressed in the heart, lung, stomach, and liver tissues of the rabbit. From immunohistochemical studies of NADase, high concentrations in both human and rabbit tissues were found in hepatocytes and sinusoidal lining cells, sinus histiocytes of the lymph node, spleen and thymus, glomerular capillary endothelial cells of the kidney, cardiac muscle, endothelium of blood vessles, and erythrocytes.