Localization of cholinesterase in Pseudomonas aeruginosa strain K

The inducible cholinesterase of Pseudomonas aeruginosa strain K (ATCC 25102) degraded propionylcholine, acetylthiocholine, acetylcholine and acetyl-beta-methylcholine at a high rate and butyrylcholine and succinylcholine at very low rates. The localization of the enzyme in the periplasmic space was indicated by a similar rate of acetylcholine degradation by intact cells or their extracts, by release of cholinesterase together with alkaline phosphatase into the culture medium during cell growth in a low phosphate-containing medium, by liberation of cholinesterase and alkaline phosphatase during lysozyme-induced conversion of cells to spheroplasts and by freezing and thawing. Threatment of cells with diazo-7-amino-1,3-naphthalenedisulphonic acid, which inactivates surface-located enzymes, abolished most of the cholinesterase and 5'-nucleotidase activities.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Cholinesterases from Plant Tissues: II. Inhibition of Bean Cholinesterase by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride (AMO-1618)

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 μm for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 μm). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Comparative study of enzymatic activity of cholinesterases of different origin in thionaphthylacetate hydrolysis reactions

A comparative determination of kinetic parameters V and Km in the reaction of hydrolysis thionaphthylacetate and well known substrate acetylthiocholine by choline esterases from different sources was conducted. It is shown that butyrylcholine esterases hydrolyze thionaphthylacetate with velocity comparable with that of hydrolysis of acetylthiocholine, while acetylcholine esterases and propionylcholine esterases hydrolyze this substrate several times slower than acetylthiocholine. The values of Km in the reactions of hydrolysis of thionaphthylacetate for all studied cholinesterases is an order higher than for acetylthiocholine except cholinesterase of blood serum of fish. This value for the latter enzyme is practically equal.     

Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into beta-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.     

The regulation of transport of glucose, gluconate and 2-oxogluconate and of glucose catabolism in Pseudomonas aeruginosa.

1. The induction by glucose and gluconate of the transport systems and catabolic enzymes for glucose, gluconate and 2-oxogluconate was studied with Pseudomonas aeruginosa PAO1 growing in a chemostat under conditions of nitrogen limitation with citrate as the major carbon source. 2. In the presence of a residual concentration of 30mM-citrate an inflowing glucose concentration of 6-8 mM was required to induce the glucose-transport system and associated catabolic enzymes. When the glucose concentration was raised to 20mM the glucose-transport system was repressed, but the transport system for gluconate, and at higher glucose concentrations, that for 2-oxogluconate, were induced. No repression of the glucose-catabolizing enzymes occurred at the higher inflowing glucose concentrations. 3. In the presence of 30mM-citrate no marked threshold concentration was required for the induction of the gluconate-transport system by added gluconate. 4. In the presence of 30mM-citrate and various concentrations of added glucose and gluconate, the activity of the glucose-transport system accorded with the proposal that a major factor concerned in the repression of this system was the concentration of gluconate, produced extracellularly by glucose dehydrogenase. 5. This proposal was supported by chemostat experiments with mutants defective in glucose dehydrogenase. Such mutants showed no repression of the glucose-transport system by high inflowing concentrations, but with a mutant apparently defective only in glucose dehydrogenase, the addition of gluconate caused repression of the glucose-transport system. 6. Studies with the mutants showed that both glucose and gluconate can induce the enzymes of the Entner-Doudoroff system, whereas for the induction of the gluconate-transport system glucose must be converted into gluconate.     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.     

Cholinesterase,acid phosphatase,and phospholipase C ofPseudomonas aeruginosa under hyperosmotic conditions in a high-phosphate medium

The presence of low choline or betaine concentrations in a culture medium containing succinate, NH₄Cl, and inorganic phosphate (Pi) as the carbon, nitrogen, and phosphate sources, respectively, permits the growth ofPseudomonas aeruginosa in a hyperosmolar medium. Dimethylglycine, acetylcholine, and phosphorylcholine were less effective as osmoprotectants than choline or betaine. Other alkylammonium compounds tested were virtually ineffective in this capacity. Bacterial growth was also observed in a hyperosmolar medium when choline was the sole carbon and nitrogen source. Choline could act as an osmoprotectant under all the conditions tested. However, the production of cholinesterase (ChE), acid phosphatase (Ac. Pase) and phospholipase C (PLC) took place only when choline was the carbon and nitrogen source. This fact confirms that the synthesis of PLC may occur even in the presence of a high Pi concentration in the medium. Inasmuch as in a high-P_i medium the synthesis of PLC and Ac. Pase (phosphorylcholine phosphatase) is dependent only on choline metabolism, it is postulated that both enzymes are involved in a set of reactions coordinated to produce the breakdown of the membrane phospholipids of the host cell in a hyperosmotic medium.     

Pathogenicity ofPseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase,acid phosphatase,and phospholipase C

The increase of cholinesterase (ChE), acid phosphatase (Ac.Pase), and phospholipase C (PLC) activities byPseudomonas aeruginosa was associated with the choline consumption in growth media of varied composition (high or low P_i concentrations, presence or absence of ammonium ion, amino acids, polyamines, peptone, or tricarboxylic acid cycle intermediates). The highest production of the three enzymes occurred in the late stationary growth phase. The simultaneous presence of alkaline phosphatase (Alk.Pase) and the above enzymes was noted when the bacteria were grown in low P_i medium plus choline, in the absence of a preferred carbon source. The importance of choline in the production of ChE, Ac.Pase, and PLC was observed in either clinical isolates or collection strains ofP. aeruginosa. These enzymes catalyze the hydrolysis of acetylcholine, phosphorylcholine, and phosphatidylcholine. Through their action the bacteria may break down various compounds (e.g., acetylcholine, from the corneal epithelium; lung surfactant dipalmitoylphosphatidylcholine; phosphorylcholine, a product of the PLC action) or cell membranes through the coordinated action of PLC and Ac.Pase or Alk.Pase. The final consequence of the action of these enzymes is an increase of the free choline concentration. Extrapolated to an in vivo situation, if the stationary growth phase resembles the conditions thatP. aeruginosa encounters in its natural environments, then it is possible to include choline among the factors promoting the pathogenicity of this bacterium.     

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa.

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.     

Quantitation of avian spermatozoan motility: Neurochemical regulation

The hypothesis that motility of avian sperm is regulated by acetylcholine was examined by treating rooster (Gallus domesticus) sperm with choline analogs and paraoxon, an inhibitor of colonesterases. Acetylcholine chloride (AChCl) was most effective, acetylthiocholine iodide and butyrylthiocholine iodide were less effective, and choline chloride was ineffective in stimulating sperm motility. Histochemical localization of cholinesterase activity with the electron microscope showed enzyme activity to be associated with membranes of the head and within fibrillar components of the tail. Increasing concentrations of paraoxon decreased cholinesterase activity and increased sperm motility. The data provide evidence that the motility of avian sperm, like that of mammal and sea urchins, may be regulated in part by a system with similarities to the cholinergic neurotransmitter system.     

Influence of Carbon or Nitrogen Starvation on Amino Acid Transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa was shown to utilize the majority of commonly occurring amino acids for growth as either the sole carbon or the sole nitrogen source. During carbon or nitrogen deprivation, the rates of transport of most of the amino acids remained unchanged; however, the transport rates for glutamate, alanine, and glycine increased under these conditions and the transport rates for leucine and valine decreased. Normal transport rates for these amino acids were resumed immediately upon the addition of the required nutrient. In the absence of an external source of carbon or of nitrogen, pool amino acids underwent rapid degradation. (14)C-Amino acid pulse experiments indicated that the constitutive amino acid catabolic enzymes, normally present in the organism during growth with glucose as the carbon source, were responsible for rapid pool losses. Nutrient starvation in the presence of chloramphenicol did not prevent amino acid catabolism. This enzymic activity is interpreted as providing P. aeruginosa with a selective advantage for survival during conditions of carbon or nitrogen starvation.     

Effect of salt composition of the medium and ethanol on cholinesterase hydrolysis of various substrates

Sodium chloride, phosphate buffer and ethanol were studied for their effect on butyryl cholinesterase hydrolysis rate of acetylcholine, acetylthiocholine, butyrylthiocholine and nonion substrate of indophenylacetate. The concentrations of 1.10(-2) = 1.10(-1) M of sodium chloride activated enzymatic hydrolysis of ion substrates at the concentrations lower than 1.10(-4) M but sodium chloride is a competitive inhibitor at higher concentrations. Phosphate buffer also activates substrates enzyme hydrolysis at the concentrations of 2.10(-4) M and lower, but it inhibits incompetitively the nonion substrate indophenylacetate hydrolysis. Ethanol activates butyrylthiocholine hydrolysis and is a competitive inhibitor in acetylthiocholine and indophenylacetate hydrolysis. The observed effects are discussed on the assumption of two forms of butyrylcholinesterase E' and E" existence. These two forms are determined by different kinetic parameters and are in equilibrium.     

Cholinesterases from plant tissues. VI. Preliminary characterization of enzymes from Solanum melongena L. and Zea mays L.

Enzymes capable of hydrolyzing esters of thiocholine have been assayed in extracts of Solanum melongena L. (eggplant) and Zea Mays L. (corn). The enzymes from both species are inhibited by the anti-cholinesterases neostigmine, physostigmine, and 284c51 and by AMO-1618, a plant growth retardant and they both have pH optima near pH 8.0. The enzyme from eggplant is maximally active at a substrate concentration of 0.15 mM acetylthiocholine and is inhibited at higher substrate concentrations. On the basis of this last property, the magnitude of inhibition by the various inhibitors, and the substrate specificity, we conclude that the enzyme from eggplant, but not that from corn, is a cholinesterase.     

Effects of bile salts on the plasma membranes of isolated rat hepatocytes.

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.     

A comparative study of acquired amidase activity in Pseudomonas species

Pseudomonas putida PP3 carrying dehalogenases I and II and Pseudomonas aeruginosa PAU3 carrying dehalogenase I coded for by plasmid pUU2 were able to grow on 2-monochloropropionic acid (2MCPA). Neither strain utilized 2-chloropropionamide (2CPA) as a carbon or nitrogen source for growth. Mutations in both strains to 2Cpa+ phenotypes (designated P. putida PPW3 and P. aeruginosa PAU5, respectively) involved the expression of an acquired 2CPA-amidase activity. The amidase followed by dehalogenase reactions in these strains constituted a novel metabolic pathway for growth on 2CPA. P. putida PPW3 synthesized a constitutive amidase of molecular mass 59 kDa consisting of two identical subunits of 29 kDa. For those amides tested this acquired enzyme was most active against chlorinated aliphatic amides, although substrate affinities (Km) and maximum rates of activity (Vmax) were poor. P. aeruginosa PAU5 acquired a 2Cpa+ phenotype by overproducing the A-amidase normally used by this species to hydrolyse aliphatic amides. The A-amidase had only slight activity towards 2CPA. However, with constitutive synthesis the mutant grew on the chlorinated substrates. Chloroacetamide (CAA) was a toxic substrate analogue for these Pseudomonas strains. A strain resistant to CAA was isolated from P. aeruginosa PAU5 when exposed to 1-10 mM-CAA. This mutant, P. aeruginosa PAU6, synthesized an inducible A-amidase. CAA-resistance depended upon the simultaneous expression of CAA-inducible amidase and dehalogenase activities.     

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

Homogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three-fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue. Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence. Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.     

Properties and localization of N-acetylglutamate deacetylase from Pseudomonas aeruginosa

The N-acetylglutamate deacetylase (EC 3.5.1.-) from Pseudomonas aeruginosa, strain PAO1, was purified 15,000-fold to electrophoretic homogeneity. The enzyme was distinct from acetylornithinase and formylglutamate hydrolase. Its molecular weight was estimated to be 90,000 by gel filtration and by sedimentation in sucrose gradients. Electrophoresis in sodium-dodecyl sulphate gels gave a single band corresponding to a molecular weight of 44,000. N-Acetylglutamate deacetylase was L-specific and showed no peptidase activity. Among 17 N-acetyl-L-amino acids tested as substrates, N-acetyl-L-glutamine, N-acetyl-L-methionine and N-acetylglycine were hydrolysed at 20% of the rate of N-acetyl-L-glutamate whereas other N-acetyl-L-amino acids were deacetylated at a rate of less than 10%. The catalytic activity depended on Co2+. The Km of the enzyme with respect to N-acetylglutamate was 1.43 mM. Preparation of spheroplasts with lysozyme in the presence of 0.2 M-MgCl2 led to the release of 80% of the enzyme activity from the cells, indicating the periplasmic localization of N-acetylglutamate deacetylase. Its localization in the periplasmic space explains the inability of P. aeruginosa argA mutants to grow on N-acetylglutamate, which is utilized by the wild-type as a carbon and nitrogen source.