Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

Formation of N-acetylglutamate by extracts of higher plants

The enzymic synthesis of N-acetylglutamate was studied in extracts of higher plant tissues, especially in sugar beet leaves (Beta vulgaris L.). Sugar beet leaves had an enzyme that transferred the acetyl group either from acetyl-CoA or from N²-acetylornithine to glutamate. The enzyme was so unstable that special precautions were necessary for its detection and appreciable purification was impossible. The Km values were 2.5 and 0.025 mM for acetyl-CoA and N²-acetylornithine, respectively. The Km for glutamate was 23 mM with acetylornithine-glutamate transacetylase and 2.7 mM with acetyl-CoA-glutamate transacetylase. The pH optimum for acetyl-CoA-glutamate transacetylase was about 7.2 whereas that for acetylornithine-glutamate transacetylase was about 8.3. Acetylphosphate, N²-acetyl-2,4-diaminobutyrate, propionyl-CoA, and succinyl-CoA were not substrates.     

An improved method for chemical alpha-oxidation

A nonisotopic assay for acetylserotonin methyltransferase (ASMT) was devised. Melatonin, the product of the enzyme reaction, is measured fluorometrically after its reaction with o-phthaldialdehyde (OPT). The reaction of melatonin with OPT is carried out in 1 n HCl to suppress the reaction of N-acetylserotonin, the substrate of ASMT, with OPT. The mixture is gassed with nitrogen just before incubation at 60°C for 60 min in order to secure the linear relationship between the concentration of melatonin and the fluorescence intensity. This method is much simpler than the isotopic assay and also has as much high sensitivity. Moreover, in this assay the enzyme can be well saturated with S-adenosylmethionine, whereas in the isotopic assay it cannot.     

Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph,Nitrobacter agilis ATCC 14123

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M_r) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M_r values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.     

Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

Purification and properties of Acinetobacter sp. endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins

A novel endo-β-N-acetylglucosaminidase capable of acting on complex type sugar chains of glycoproteins was found in the culture broth of a bacterium which was isolated from soil and identified as Acinetobacter sp. The enzyme was purified to homogeneity on polyacrylamide gel electrophoresis by successive purification procedures involving ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G-150. Its molecular weight was about 35,000 on gel filtration. The optimum pH was 3.0–3.5, and the enzyme was stable in the pH range from 6–8. The enzyme had high activity on dansyl ovalbumin glycopeptide, and also could hydrolyze dansyl asialotransferrin glycopeptide and dansyl transferrin glycopeptide containing complex type sugar chains. The K_m value for dansyl asialotransferrin glycopeptide as the substrate of enzyme assay was 0.68 mM. The enzyme could release complex type sugar chains from intact asialotransferrin without the addition of any detergent.     

Purification and properties of phosphotransacetylase from the eucaryotic green alga Chlorogonium elongatum

Phosphotransacetylase (EC 2.3.1.8) was detected in cell-free crude extracts of starch-fermenting eucaryotic green algae. The enzyme was purified from autotrophically grown Chlorogonium elongatum. The purified enzyme fraction, after affinity chromatography, shows a single protein band upon acrylamide gel electrophoresis and has a molecular weight of 280 000. It consists of six subunits of identical molecular weight (44 000). The pH and temperature optima for the eucaryotic phosphotransacetylase are 7.6 and 28°C, respectively. The K_m values at 25°C (pH 7.6) for acetyl-CoA and phosphate are 0.078 mM and 5.440 mM, respectively, and in the reverse reaction (acetyl-CoA synthesis) for CoA and acetyl phosphate 0.093 mM and 0.310 mM, respectively. The maximum velocity of the forward reaction was 1627 nkat/mg protein and of the reverse reaction 8582 nkat/mg protein. The activity of the eucaryotic phosphotransacetylase strictly depends on the presence of univalent cations (ammonium, K_a = 9 mM; potassium, K_{a = 12.5 mM}). Inactivation studies with iodoacetamide and iodoacetic acid revealed the presence of an essential sulphhydryl group at the catalytic site. Arsenolytic and product inhibition studies indicate a rapid equilibrium random bi-bi reaction mechanism for the enzyme from C. elongatum. The control of the enzyme activity in the forward reaction by both pyruvate and NADH gives evidence for a physiological function of phosphotransacetylase in anaerobic energy metabolism of eucaryotic green algae rather than in aerobic acetate activation.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Characterization of Acyl Coenzyme A Carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis, Which Produce Multiple Methyl-Branched Mycocerosic Acids

Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin–Sepharose 4B-CL gel with d-biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 μmol min⁻¹ mg⁻¹, respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K_m values for propionyl-CoA, ATP, Mg²⁺, and NaHCO₃ were 70 μM, 100 μM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K_m was higher and the V_max was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively (S)-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n-fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.     

Purification and characterization of ferredoxin–nitrite reductase from the eukaryotic microalga Monoraphidium braunii

Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg^–1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate K_m values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii.     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca 1 b

Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 μmoles H₂/min·mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5–10 μM concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH₂. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the K _m was 0.063 mM H₂ and for saturating hydrogen concentration the K _m was 0.123 mM NAD.     

A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: Purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a #x003B2;-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 °C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. #x003B2;-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 M and 25 M for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of #x003B2;-ketothiolase. (Mol Cell Biochem 269: 27–36, 2005)     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-l-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.     

Partial purification and characterization of 3-hydroxy-3-methylglutaryl coenzyme a reductase from the leaves of guayule (Parthenium argentatum)

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been isolated and was partially purified from the leaves of Parthenium argentatum. The enzyme was found to be associated both with the cytosol and the chloroplasts. Ten mM dithiothreitol was essential to prevent loss of activity. Optimum activities of cytosolic and chloroplastic fractions were observed at pH 7.0 and 7.5 respectively. Preincubation of the reaction mixtures with CoA, acetyl-CoA, σ-phenanthroline and iodoacetamide resulted in the progressive loss of enzyme activity. 3-Hydroxybutyrate and mevalonate also inhibited the enzyme. The Michaelis constants of the enzyme for HMG-CoA and NADPH were 0.25 and 0.31 mM respectively for the cytosolic enzyme, while those for the chloroplastic enzyme were 0.018 and 0.42 mM respectively. Inhibition studies indicated that hydroxybutyrate was a competitive inhibitor with respect to HMG-CoA. The inhibition of mevalonate was competitive with HMG-CoA and non-competitive with NADPH.     

Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5′AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1–3 μM and stoichiometry of 2 mol mol⁻¹ enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol⁻¹ enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol⁻¹ enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme–acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 μM Mg²⁺ ions. The velocity at pH 7.0 with Mg²⁺ exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol⁻¹ enzyme, indicating that Mg²⁺ ions increased the rate of decomposition of the enzyme–acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.     

Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents

Thiolase (acetyl-coenzyme A [CoA] acetyltransferase, E.C. 2.3.1.19) from Clostridium acetobutylicum ATCC 824 has been purified 70-fold to homogeneity. Unlike the thiolase in Clostridium pasteurianum, this thiolase has high relative activity throughout the physiological range of internal pH of 5.5 to 7.0, indicating that change in internal pH during acid production is not an important factor in the regulation of this thiolase. In the condensation direction, the thiolase is inhibited by micromolar levels of CoA, and this may be an important factor in modulating the net condensation of acetyl-CoA to acetoacetyl-CoA. Other cofactors and metabolites that were tested and shown to be inhibitors are ATP and butyryl-CoA. The native enzyme consists of four 44,000-molecular-weight subunits. The kinetic binding mechanism is ping-pong. The K_m value for acetyl-CoA is 0.27 mM at 30°C and pH 7.4. The K_m values for sulfhydryl-CoA and acetoacetyl-CoA are, respectively, 0.0048 and 0.032 mM at 30°C and pH 8.0. The active site apparently contains a sulfhydryl group, but unlike other thiolases, this thiolase is relatively stable in the presence of 5,5′-dithiobis(2-nitrobenzoic acid). Studies of thiolase specific activity under various types of continuous fermentations show that regulation of this enzyme at both the genetic and enzyme levels is important.     

Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract—

• 1 Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation-exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G-200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01-0-3 μmol acetylcholine formed per min per mg protein.
• 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000.
• 3 In the catalysed reactions, , k₁, was about four times k₂, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0-75 mM; acetyl-CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0-75-5 MM; CoA = 25-150 μM).
• 4 CoA inhibited acetylcholine synthesis by competing with acetyl-CoA (K₁, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl-CoA. These data indicate an ordered reaction mechanism; acetyl-CoA probably always binds before choline.