Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

Eder-Colli, Lorenza; Amato, Stefano

doi:10.1016/0306-4522(85)90235-0

Cited by 45 publications

(19 citation statements)

References 33 publications

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“…Eckenstein et al (21,22) purified porcine brain ChoAcTase using a procedure that selected for soluble proteins. However, several biochemical experiments suggest that the enzyme also exists in a membrane-bound form, with a distinct isoelectric point and molecular weight (35,36), although this apparent heterogeneity of ChoAcTase may be due to interactions with proteins or cleavage by proteolytic enzymes (37,38). Analysis of the hydropathy profile and secondary structure predicted from the pChAT-1 sequence displayed in Fig.…”

Section: Discussionmentioning

confidence: 99%

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

Berrard

Brice

Lottspeich

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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(3, 4). The analysis, in molecular terms, of the mechanisms underlying this plasticity requires the study of the genes encoding these two enzymes.We have identified (5-7) cDNA clones corresponding to rat and human tyrosine hydroxylases. Here, we describe the isolation of a cDNA clone-pChAT-1-that encodes an active porcine ChoAcTase enzyme. The nucleotide and complete amino acid sequence is reported. ¶ Some structural characteristics of porcine ChoAcTase are discussed, and the sequence is compared with that of Drosophila melanogaster reported by Itoh et al. (8). MATERIALS AND METHODSConstruction of a Randomly Primed cDNA Library in the XgtlO Vector. Total RNA from porcine ventral spinal cord was extracted as described by Lomedico and Saunders (9). Poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. Random DNA sequences 20-50 nucleotides in length were prepared by sonication and DNase I digestion of calf thymus DNA (10) and used as primers for cDNA synthesis. First-strand cDNA was synthesized from 2.5 ,g of ventral spinal cord poly(A)+ RNA with 30-fold excess of random primer. The second-strand synthesis and following steps were carried out using standard procedures (11, 12). The longest cDNAs [_500 base pairs (bp)] were selected on a 5-20o (wt/vol) sucrose gradient and ligated to the XgtlO vector. The amplified library contained =1.2 x 106 independent recombinant phages.Oligonucleotide Screening. The N-terminal sequence of porcine brain ChoAcTase was determined as described (13). A mixture of oligodeoxynucleotides, each containing eight different chains of 29 nucleotides, was prepared with a Biosearch DNA synthesizer model 8600 by the phosphoramidite method and purified by PAGE. The probes were end-labeled to a minimal specific activity of 8 x 108 cpm/,ug. About 106 recombinant phages were plated at 50,000-70,000 plaques per 13-cm plate, and duplicate filters were prepared. Filters were hybridized at 35°C with oligonucleotides in 6x SSC (lx SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 5x Denhardt's solution (lx Denhardt's solution = 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.05% sodium pyrophosphate, herring sperm DNA at 0.1 mg/ml, and Escherichia coli tRNA at 0.1 mg/ml. Filters were then washed at 35°C, 40°C, and 45°C in 6x SSC containing 0.05% NaDodSO4. Positive clones were isolated after three successive rounds of screening. Phage DNA was prepared as Abbreviations: ChoAcTase, choline acetyltransferase; NVP, 4-(1-naphthylvinyl)pyridine. §To whom reprint requests should be addressed.

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Section: Discussionmentioning

confidence: 99%

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

Berrard

Brice

Lottspeich

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Numerous reports in the literature indicate that ChAT is predominantly a cytosolic protein but that some fraction of the total neuronal enzyme associates both ionically and nonionically with plasma membrane (13). The proportion of enzyme that is membrane-bound appears to vary between species and at different stages of development (14,15).…”

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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“…The presence of a membrane form of ChAT has been reinvestigated, and a fraction that is high-salt insoluble and detergent extractable has been reported in Torpedo, rat, mouse, and human (Benishin and Carroll, 1983;Eder-Colli and Amato, 1985). Three putative membrane-associated forms of ChAT have been characterized on the basis of their elution from membranes with (a) 100 mM sodium phosphate, (b) 500 mM NaC1, and (c) detergent (Benishin and Carroll, 1983;Barochovsky et al, 1988).…”

Section: Intracellular Localization Of Chatmentioning

confidence: 99%

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Hersh

1994

Journal of Neurochemistry

152

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It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/ translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein‐protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic‐specific elements and the characterization of the trans‐acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three‐dimensional structure of the enzyme.

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Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

Cited by 45 publications

References 33 publications

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

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

Choline Acetyltransferase: Celebrating Its Fiftieth Year

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