Cholesterol esterase catalyzed hydrolysis of mixed micellar thiophosphatidylcholines: a possible charge-relay mechanism

Mechanistic features of cholesterol esterase catalyzed hydrolysis of two thiophospholipids, rac-1-(hexanoylthio)-2-hexanoyl-3-glycerophosphorylcholine (6TPC) and rac-1-(decanoylthio)-2-decano-yl-3-glycerophosphorylcholine (10TPC), have been characterized. The hydrolysis of 10TPC that is contained in mixed micelles with Triton X-100 occurs strictly at the micellar interface, since the reaction rate is independent of the micelle concentration but depends hyperbolically on the mole fraction of the substrate in the micelles. This latter observation allows one to calculate the interfacial kinetic parameters V*max and K*m. The hydrolyses of 10TPC and p-nitrophenyl butyrate are similarly inhibited by the transition state analogue inhibitor phenyl-n-butylborinic acid, and therefore, physiological and nonphysiological substrates are processed at the same active site. The similarity of k*cat values for the acyl-similar substrates 10TPC and p-nitrophenyl decanoate indicates that the phospholipase A1 activity of cholesterol esterase is partially rate limited by turnover of a decanoyl-enzyme intermediate. Solvent isotope effects on V*max and V*max/K*m (which monitors acylation only) are approximately 2-3 and are consistent with transition states that are stabilized by general acid-base proton transfers. Proton inventories of V*max/K*m indicate that simultaneous proton transfers stabilize the acylation transition state, which requires a multifunctional acid-base machinery (perhaps a charge-relay system) in the cholesterol esterase active site. Similar results are obtained for the 6TPC reaction, both in the presence and absence of Triton X-100 micelles.     

Characterization of d-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid

The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.     

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.     

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine, a highly specific chromogenic substrate of phospholipase A2

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine (2-thioPC), a structurally modified phospholipid analog is specifically hydrolyzed by phospholipase A2 to liberate 2-thiolysophosphatidylcholine and palmitic acid. The sulfhydryl group of the product is readily trapped by 5,5'-dithiobis (2-nitrobenzoic acid) allowing continuous spectrophotometric monitoring of the enzymatic reaction. The rates of hydrolysis by bee-venom phospholipase A2 have been determined in a series of Triton X-100 containing mixed micelles. At 1 mM 2-thioPC increasing the concentration of Triton X-100 from 4 to 16 mM changes the specific activity of bee-venom phospholipase A2 from 96.9 to 17.9 mumol/min/mg, about one order of magnitude lower than dipalmitoyl phosphatidylcholine hydrolysis in micelles of similar composition. The chromogenic substrate is the first phospholipid analog exhibiting absolute specificity for phospholipase A2 and should be applicable to spectrophotometric detection and kinetic characterization of both water soluble and membrane-bound forms.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

Characteristics of phospholipase D in reverse micelles of Triton X-100 and phosphatidylcholine in diethyl ether

Summary Phospholipase D, from cabbage, is active in reverse micelles formed from its substrate phosphatidylcholine and Triton X-100 in diethyl ether. The activity is optimum at w₀=12.5. The increase of the molar ratio of Triton X-100/substrate from 1:4 to 2:1 results in an activity decrease by 25 %. At 136 mM Triton X-100 the K_M value in reverse micelles is 136 mM, whereas it is 0.40 mM in the aqueous system containing SDS.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl2 and DIDS. The IC50 for HgCl2 was 0.16+/-0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9+/-0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

Characterization of an exceedingly active NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima

An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80 degrees C. The V(max) was 230 +/- 14 mumol/min/mg (k(cat)/K(m) = 548,000 min(-1) mM(-1)), and the K(m) values for NADH and oxygen were 42 +/- 3 and 43 +/- 4 muM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.     

Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea)

beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.     

Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis

Extracellular Penicillium fellutanum exo-beta-D-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP(2)GM(ii) and pP(25)GM(ii) (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-beta-D-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP(2)GM(ii) or pP(25)GM(ii). The k(cat)/K(m) value for pP(25)GM(ii) is 1.7 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 10(5) M(-1) s(-1), respectively. Variability in the k(cat)/K(m) values is due primarily to differences in K(m) values; the k(-1)/k(1) ratio likely provides the most influence on K(m). k(cat) increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP(x)GM(ii) added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml(-1)h(-1). No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP(x)GM(ii) and of related polymers is proportional to the k(cat)/K(m) value of each polymer. The kinetic data show that the k(cat)/K(m) value increases as the number of phosphodiesters of pP(x)GM(ii) decreases, also resulting in an increase in the activity of exo-beta-D-galactofuranosidase.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).     

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.     

Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes.

We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The Km for the surface concentration of phosphatidic acid was 0.30 mol%. The enzyme was inhibited by sphingosine and chloropromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.     

Glycosyl-phosphatidylinositol anchored acetylcholinesterase as substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

We investigated the enzymatic properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus towards glycosyl-phosphatidylinositol anchored acetylcholinesterase (AChE) from bovine erythrocytes and Torpedo electric organ as substrate. The conversion of membrane from AChE to soluble AChE by PI-PLC depended on the presence of a detergent and of phosphatidylcholine. In presence of mixed micelles containing Triton X-100 (0.05%) and phosphatidylcholine (0.5 mg/ml) the rate of AChE conversion was about 3 times higher than in presence of Triton X-100 alone. Furthermore, inhibition of PI-PLC occurring at Triton X-100 concentrations higher than 0.01% could be prevented by addition of phosphatidylcholine. Ca2+, Mg2+ and sodium chloride had no effect on PI-PLC activity in presence of phosphatidylcholine and Triton X-100, whereas in presence of Triton X-100 alone sodium chloride largely increased the rate of AChE conversion. Determination of kinetic parameters with three different substrates gave Km-values of 7 microM, 17 microM and 2 mM and Vmax-values of 0.095 microM.min-1, 0.325 microM.min-1 and 56 microM.min-1 for Torpedo AChE, bovine erythrocyte AChE and phosphatidylinositol, respectively. The low Km-values for both forms of AChE indicated that PI-PLC not only recognized the phosphatidylinositol moiety of the anchor but also other components thereof.