The proportion of amphiphilic choline acetyltransferase inDrosophila melanogaster is higher than in rat orTorpedo and is developmentally regulated

Salem, Natalie; Medilanski, Jana; Pellegrinelli, Nathalie; Eder-Colli, Lorenza

doi:10.1016/0006-8993(93)90876-o

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…The proportion of enzyme that is membrane-bound appears to vary between species and at different stages of development (14,15). The means by which ChAT binds nonionically to membranes has not been elucidated; it does not have obvious hydrophobic domains for association with the lipid bilayer, and the presence of covalent modifications such as a glycosylphosphatidylinositol linkage have been controversial (16 -18).…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Dobránsky

Davis

Rylett

2001

Journal of Biological Chemistry

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Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and sitedirected mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time-and dose-related increases in specific activity of the enzyme; in control and phorbol estertreated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [ 32 P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.Choline acetyltransferase (ChAT, 1 EC 2.3.1.6) catalyzes synthesis of the neurotransmitter acetylcholine (ACh) in cholinergic neurons in peripheral and central nervous systems. These neurons control a wide range of physiological and biochemical processes in most organ systems, including regulation of cardiovascular and motor functions, and cognitive functions such as learning, attention, and memory. Diminished ChAT activity signals degeneration of cholinergic neurons in a number of neurodegenerative disorders. For example, a consistent finding in necropsy brain of subjects with Alzheimer disease is profound loss of ChAT that correlates with diminished cognitive function early in the course of the disease. Decreased ChAT activity can be accounted for, at least in part, by loss of cholinergic neurons, but may also be related to decreased expression of cholinergic phenotypic genes and/or altered regulation of the enzymes catalytic activity leading to decreased function.There is polymorphism in expression of mRNA for ChAT and, in human only, one of these transcripts, denoted the M isoform, has two translation initiation sites yielding proteins with apparent molecular masses of 69 and 82 kDa; all other transcript isoforms encode the 69-kDa form of enzyme only (1, 2). We demonstrated recently that the 82-kDa form of the enzyme is targeted to nucleus of cells, whereas 69-kDa ChAT is local...

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Dobránsky

Davis

Rylett

2001

Journal of Biological Chemistry

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“…This association partly depends on a reversible binding that changes according to the ionic milieu (Fonnum, 1968;Fonnum and Malthe-Sørenssen, 1973) and might be controlled by the phosphorylation state of ChAT (Bruce and Hersh, 1989;Schmidt and Rylett, 1993a). Also, a fraction of cellular ChAT is now considered membranous (see, e.g., Smith and Carroll, 1980;Eder-Colli and Amato, 1985;Peng et al, 1986;Salem et al, 1993;Schmidt and Rylett, 1993b); it may be inserted into membranes, removable by proteinase K (Eder-Colli et al, 1992), or held there by a glycosylphosphatidylinositol link (Carroll and Smith, 1990;Smith and Carroll, 1993). The microtubule-dependent transport of ChAT could result from one or both of these membrane associations.…”

Section: Maintenance Of Chat Activity In Nerve Terminalsmentioning

confidence: 99%

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Tandon¹,

Bachoo

Weldon

et al. 1996

Journal of Neurochemistry

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These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine‐treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2‐Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2‐Hz stimulation of colchicine‐treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20‐Hz stimulation was depressed. The amount of ACh released during 5‐Hz stimulation in the presence of vesamicol by the terminals of colchicine‐treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine‐sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half‐life of ChAT in sympathetic preganglionic terminals is ∼4 days. (c) One consequence of colchicine‐induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.

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“…We have already shown that in the central nervous system of the fly D. melanogaster, ChAT activity exists in two molecular forms: a soluble hydrophilic form and a membrane-bound amphiphilic form (Salem et al, 1993). This was established by using sequential solubilization and Triton X-114 phase separation; a method that allows the separation of amphiphilic from hydrophilic proteins (Bordier, 1981).…”

Section: Introductionmentioning

confidence: 99%

Hydrophilic and Amphiphilic Forms of Drosophila Choline Acetyltransferase are Encoded by a Single mRNA

Salem¹,

Medilanski²,

Pellegrinelli³

et al. 1994

Eur J of Neuroscience

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We have previously shown that the enzyme choline-O-acetyltransferase (ChAT) exists in a hydrophilic and an amphiphilic form in Drosophila head. A complementary DNA clone of 4.2 kb containing the entire coding region of ChAT was isolated from a cDNA library of Drosophila heads. The cDNA was subcloned in an expression vector and injected into the nucleus of Xenopus oocytes. Injected oocytes expressed high levels of ChAT activity. This activity was inhibited by bromoacetylcholine, a specific inhibitor of the enzyme. In the present study the non-ionic detergent Triton X-114 was used to analyse whether the expression of hydrophilic and amphiphilic ChAT was or was not directed by a single cDNA. The two forms of ChAT were found to be synthesized in injected oocytes. Approximately 9% of the recombinant enzyme partitioned as amphiphilic activity. This value was similar to that found for native amphiphilic ChAT in Drosophila heads. Sedimentation in sucrose gradients of amphiphilic enzyme was found to be influenced by the type of detergent present in the gradient whereas this was not the case for hydrophilic ChAT. Hydrophilic and amphiphilic enzyme activities differed in some of their biochemical properties. Amphiphilic ChAT was less sensitive to inhibition by the product acetylcholine than was hydrophilic ChAT. Moreover, amphiphilic ChAT was found to be more resistant than hydrophilic ChAT to heat inactivation at 45 degrees C. These properties were observed for the native as well as for recombinant ChAT. These results demonstrate that the hydrophilic and amphiphilic forms of ChAT are derived from one mRNA.

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The proportion of amphiphilic choline acetyltransferase inDrosophila melanogaster is higher than in rat orTorpedo and is developmentally regulated

Cited by 7 publications

References 27 publications

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Hydrophilic and Amphiphilic Forms of Drosophila Choline Acetyltransferase are Encoded by a Single mRNA

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