Critical Active-Site Residues Identified by Site-Directed Mutagenesis in Pseudomonas aeruginosa Phosphorylcholine Phosphatase, A New Member of the Haloacid Dehalogenases Hydrolase Superfamily

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg²⁺, PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues ³¹DMDNT³⁵, ¹⁶⁶SAA¹⁶⁸, and K²⁴²/²⁶¹GDTPDSD²⁶⁷, respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K_m1 0.03 mM, K_m2 0.5 mM) or p-NPP (K_m 2.1 mM). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.     

INHIBITION OF BACTERIAL AND MAMMALIAN CHOLINE ACETYLTRANSFERASES BY STYRYLPYRIDINE ANALOGUES

—Choline acetyltransferase was extracted from Lactobacillus plantarum by relatively gentle procedures involving penicillin treatment, osmotic shock and passage through a French pressure cell. After partial purification, the extract was compared with choline acetyltransferase of calf caudate nucleus for kinetic properties and response to a class of inhibitors which consists of analogues of styrylpyridine. Both enzymes obeyed a sequential mechanism with Michaelis constants for the bacterial enzyme, K_m= 8 μm vs. acetyl-CoA and 0·44 mm vs. choline; and for the caudate nucleus enzyme, K_m= 15 μm vs. acetyl-CoA and 0·8 mm vs. choline. Both were stabilized by dithiothreitol and EDTA. The extracts differed in that the bacterial enzyme was more labile and apparently was susceptible to conformational changes, which modified its response to the styrylpyridinetype inhibitors. The use of intact cells of Lactobacillus plantarum as an in vivo system for studying the inhibition of choline acetyltransferase by styrylpyridines was possible only for non-quaternary analogues, which exist as an equilibrium mixture of charged and uncharged species.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

The synthesis of choline and ethanolamine phosphoglycerides in neuronal and glial cells of rabbit in vitro

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine-5′-phosphate choline (cytidine-5′-phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell-enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine-5′-phosphate choline and cytidine-5′-phosphate ethanolamine, respectively. Several kinetic properties of the choline-phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor cytidine-5′-phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine-dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.     

A method for measurement of the specific radioactivity of the choline moiety of choline phospholipids.

The specific radioactivity of a choline phospholipid has been determined by a double-isotope method. Purified phospholipid was hydrolyzed to release labeled choline, and choline kinase was employed to label the choline with ³²P from [γ-³²P]ATP. The double-labeled phosphorylcholine was purified by ion-exchange chromatography on QAE-Sephadex, and the specific radioactivity of the choline was calculated from the isotope ratio. The method is sensitive, requiring only 5 nmol of choline with a specific radioactivity of 1 μCl/μmol, and the chromatographic isolation of phosphorylcholine is simple and reproducible.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Ethanolamine kinase from Culex pipiens fatigans

Ethanolamine kinase was partially purified from the larvae of Culex pipiens fatigans and its properties were studied. The enzyme was separated from choline kinase by acetic acid precipitation at pH 5.0 of a 13,000g supernatant of the larval homogenate. Alkaline phosphatase activity was removed from the enzyme preparation by the acid treatment followed by ammonium sulfate fractionation. The enzyme was localized in the cytosolic fraction and had a requirement for Mg²⁺ as a cofactor. The K_m values for ethanolamine and ATP were 4 × 10^?4 and 1.54 × 10^?4m, respectively. The affinity of the enzyme for nucleotide triphosphates was in the order, ATP > ITP > GTP while UTP and CTP were poorly utilized. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme activity and reduced glutathione protected the enzyme from their inhibition. Choline and serine had no effect on the enzyme activity. The enzyme had a molecular weight of 44, 000 daltons as determined by gel filtration chromatography. Eggs contained the highest specific activity of the enzyme while adult insects had the highest total enzyme activity.     

Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

Modification of the (Na+ + K+)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na⁺ + K⁺)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl (k_0.5 ca. 0.6 mm) or ATP (K_0.5 ca. 1 μm) in the medium protected against inactivation, whereas MgCl₂ (k_0.5 ca. 1 mm) increased inactivation. K⁺-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na⁺-dependent phosphorylation of the enzyme and Na⁺-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k_0.5 for K⁺-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k_0.5 for neither K⁺ nor Na⁺ was increased, although the apparent k_m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K⁺ at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k_m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a pK_a for the reactive enzyme groups of 7.5–8; and TNBS affected K⁺-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K⁺ at these sites.     

Amino acid sequence at the phosphorylated site of rat liver fructose-1,6-diphosphatase and phosphorylation of a corresponding synthetic peptide.

Rat liver fructose-1,6-diphosphatase was phosphorylated with (³²P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(³²P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent K_m of 400 μM, while the apparent K_m value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The V_max value was 20 times higher for the peptide than for the enzyme.     

Properties of Purified L-Ornithine Decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO₂mdh^-1mdmg^-1. The purified enzyme was rather stable at 37°C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the K_m for the substrate L-ornithine was 0.11 mM, and the K_m for the cofactor pyridoxal 5-phosphate was 0.12 μM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated K_i of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a K_i of 1.6 mM and by α-difluoromethylornithine with a K_i of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate. Polyanions were activators of the enzyme: The half maximal ODC stimulating concentrations were 2.2 μgmdml^-1 for RNA, 6.1 μgmdml^-1 for DNA, and 0.25 μgmdml^-1 for heparin. These results indicate that ODC from T. thermophila shares several properties with ODC preparations from other organisms but in some respects, especially in activator and inhibitor specificity, there are some special qualities unique to this particular protozoan enzyme.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

OPOSSUM KIDNEY CELLS TAKE UP L-DOPA THROUGH AN ORGANIC CATION POTENTIAL-DEPENDENT AND PROTON-INDEPENDENT TRANSPORTER

The present work was aimed at studying the kinetics and nature of the l-DOPA transporter in opossum kidney (OK) cells. Saturation experiments were performed in OK cells incubated for 6 min with increasing concentrations of l-DOPA (10 to 2500 μm); non-linear analysis of the saturation curve revealed for l-DOPA aK_mof 129 μm (114, 145) and aV_maxof 30.0±0.4 nmol mg protein^?16 min^?1The uptake of l-DOPA (250 μm) was inhibited in a concentration-dependent manner by cyanine 863, an organic cation inhibitor, with aK_ivalue of 638 (430, 947) μmthe organic anion inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), was devoid of effect upon the uptake of l-DOPA. The uptake of l-DOPA (250 μm) was significantly (P<0.02) decreased (25% reduction) when cells were incubated in the presence of 137 mm K⁺plus 5 mm Na⁺when compared with the control condition (137 mm Na⁺plus 5 mm K⁺); substitution of NaCl by choline chloride (137 mm) did not affect l-DOPA uptake. Similarly, inwardly or outwardly directed proton gradients of 0.5 pH units (7.9, 7.4, 6.9, 6.4 and 5.9) were found not to change l-DOPA uptake. In conclusion, the l-DOPA uptake system in OK cells has the characteristics of an organic cation potential-dependent and proton-independent transporter.     

Acetylcholine Synthesizing Enzymes in Frog Skeletal Muscle

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (V_max, 2 nmol g¹ h^?1, K_mfor choline, approx. 50 μ, m ). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4-(1-naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine-synthesizing activity of a different enzyme not saturated by 10 mm -choline. The non-specific enzyme was present at and away from the endplate area, and it was not affected by denervation.