The 'compartmentation' of choline acetyltransferase within the synaptosome.
ABSTRACT: 1. Choline acetyltransferase may be isolated in either a bound or soluble form after hypo-osmotic treatment of a crude synaptosome fraction, depending on the conditions. 2. In the bound form, the enzyme appears to be associated with the larger membrane fragments rather than with synaptic vesicles. 3. The bound form is predominant at slightly acid pH values and low ionic strength, the soluble form under more physiological conditions of pH and ionic strength. 4. Sodium chloride, potassium chloride, magnesium chloride and calcium chloride at similar ionic strengths solubilize the enzyme. 5. Choline acetyltransferase was found to be soluble under these conditions after release from synaptosomes from rat and pigeon cerebra, guinea-pig cortex and rabbit cortex, caudate nuclei, diencephalon and midbrain. 6. Certain isoenzymes of lactate dehydrogenase behaved similarly.
Project description:1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.
Project description:1. Michaelis constants for human placental choline acetyltransferase were shown to be dependent on the concentration of the second substrate present. The primary plots indicate a sequential rather than a Ping Pong mechanism and are of the same type with 300mm- and 500mm-sodium chloride. 2. Similar results have been obtained with rabbit brain choline acetyltransferase. 3. Product inhibition of the forward reaction has been studied. CoA inhibits competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibits competitively with respect to choline and non-competitively with respect to acetyl-CoA. No inhibition is given by acetylcholine when the enzyme is saturated with choline. 4. It is concluded that human placental choline acetyltransferase has an ordered mechanism of the Theorell-Chance type.
Project description:Six ionic liquid (IL)-forming ions (choline, tetrabutylphosphonium, tetrabutylammonium, and trimethylhexadecylammonium cations, and chloride and docusate anions) were paired with acyclovir as the counterion to form four low melting solid salts and two waxes; five of these compounds could be classified as ILs. All of the newly synthesized acyclovir ILs exhibited increased aqueous solubilities by at least 2 orders of magnitude when compared to that of neutral acyclovir. For three of the prepared compounds, the solubilities in simulated body fluids (phosphate-buffered saline, simulated gastric, and simulated intestinal fluids) were also greatly enhanced when compared to that of neutral acyclovir. Acyclovir in its anionic form was more water- or buffer-soluble than acyclovir in its cationic form, though this might be the effect of the particular ions, indicating that the solubilities can be finely tuned by proper choice of the cationic or anionic form of acyclovir and the counterion paired with it.
Project description:1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(-)-carnitine plus CoA or of bromoacetyl-CoA plus (-)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromoacetyl substrate analogue; in each case about 1mole of inhibitor is required to inactivate completely 1mole of enzyme of molecular weight 58000+/-3000. 3. Inhibition by bromoacetyl-CoA plus (-)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (-)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (-)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4 degrees . 5. Incubation with S-carboxymethyl-CoA (-)-carnitine ester causes a slow inhibition of carnitine acetyltransferase. 6. The formation of bound S-carboxymethyl-CoA (-)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme-substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase.
Project description:1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3-7.6, 7.7-7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent K(m) for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.
Project description:Ionic media comprising 1-butyl-3-methylimidazolium chloride and the acidic deep eutectic solvent choline chloride/oxalic acid as co-solvent-catalyst, very efficiently convert various cellulosic substrates, including native cellulosic biomass, into water-soluble carbohydrates. The optimum reaction systems yield a narrow range of low molecular weight carbohydrates directly from cellulose, lignocellulose, or algal saccharides, in high yields and selectivities up to 98?%. Cellulose possesses significant potential as a renewable platform from which to generate large volumes of green replacements to many petrochemical products. Within this goal, the production of low molecular weight saccharides from cellulosic substances is the key to success. Native cellulose and lignocellulosic feedstocks are less accessible for such transformations and depolymerisation of polysaccharides remains a primary challenge to be overcome. In this study, we identify the catalytic activity associated with selected deep eutectic solvents that favours the hydrolysis of polysaccharides and develop reaction conditions to improve the outcomes of desirable low molecular weight sugars. We successfully apply the chemistry to raw bulk, non-pretreated cellulosic substances.
Project description:1. Michaelis constants of goldfish brain choline acetyltransferase were found to depend on the concentration of the second substrate present and on the temperature to which the fish had been adapted. 2. Primary plots constructed from results obtained with enzyme prepared from cold-adapted or warm-adapted fish indicated that synthesis of acetylcholine took place by a sequential mechanism. 3. The affinity of choline acetyltransferase for acetyl-CoA was about 100 times that for choline irrespective of whether the enzyme had been prepared from warm-adapted or cold-adapted fish. 4. The maximum rate at which choline acetyltransferase synthesized acetylcholine and the energy of activation for this synthesis remained independent of the previous environmental temperature of the fish. 5. The affinity of choline acetyltransferase for choline and acetyl-CoA showed a complex dependence on temperature. The affinity of the enzyme from cold-adapted fish for substrates increased as the incubation temperature was lowered, whereas that of the enzyme from warm-adapted fish first increased and then decreased. 6. The maximum affinity of choline acetyltransferase for both substrates, from both cold-adapted and warm-adapted fish, occurred at temperatures that corresponded approximately to the respective environmental temperatures of the fish. 7. These changes in enzyme affinity for substrates are not thought to be due to the presence of isoenzymes. Their adaptive significance is unknown, but it could be connected with the maintenance of the enzyme in a stable form.
Project description:Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.
Project description:1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-(14)C]acetate or sodium [(3)H]acetate by incubation with CoA, ATP, Mg(2+) and extract from acetone-dried pigeon liver were used. 3. [1-(14)C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [(3)H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [(3)H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively.
Project description:1. Labelled precursors of choline, namely ethanolamine, dimethylaminoethanol and methionine and also labelled choline itself were injected intraperitoneally into the adult female rat and the incorporation into lipids and water-soluble fractions was traced in liver, blood and brain. 2. No significant free choline was detected and no labelling of the phosphorylcholine of blood. There was, however, considerable labelling of the phosphorylcholine of brain and liver. 3. After intracerebral injection, [1,2-(14)C]dimethylaminoethanol was rapidly phosphorylated and converted into phosphatidyldimethylaminoethanol, presumably by the cytidine pathway. 4. In view of the pattern of labelling and the amount of phosphatidylcholine in the tissues examined, it seems highly likely that choline is transported to the brain by the blood in a lipid-bound form.