Microdetermination of Choline Acetylase in Nervous Tissue

McCaman, Richard E.; Hunt, Judith M.

doi:10.1111/j.1471-4159.1965.tb06762.x

Cited by 364 publications

(80 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The values of ChAc activity published by McCaman and co-workers (McCaman & Hunt, 1965;Buckley et al 1967;Goldberg et al 1969) were, however, much lower (2-4,umoles of acetylcholine/hr./g. of rat brain) than those obtained with acetyl-CoA formed in 8itu (5-8,umoles of acetylcholine/hr./g.)…”

Section: Extraction Efficiencymentioning

confidence: 83%

“…The introduction of radioactively labelled substrates has provided sensitive and accurate assay methods for these enzymes. Radiochemical methods for the assay of ChAc depend on the separation of the synthesized labelled acetyleholine from radioactive substrates such as acetyl-CoA and acetate and from by-products. This has been achieved by precipitation of acetyleholine with reineckate (McCaman & Hunt, 1965), sodium tetraphenylboron (Fonnum, 1966) or periodide (Goldberg, Kaita & McCaman, 1969), by isolation of acetyleholine by electrophoresis (Potter, Glover & Saelens, 1968;Giller & Schwartz, 1968), or by removing labelled anions from acetylcholine with an anion-exchange column (Schrier & Schuster, 1967). The only method that has been applied to * Abbreviations: ChAc, choline acetyltransferase (acetylCoA-choline O-acetyltransferase, EC 2.3.1.6); AChE, acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7).…”

mentioning

confidence: 99%

“…microgram quantities of tissue is the method of McCaman & Hunt (1965), and a modification of this (Buckley, Consolo, Giacobini & MeCaman, 1967).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

Fonnum

1969

Biochemical Journal

1,039

206

View full text Add to dashboard Cite

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 [1−14C]acetate or sodium [3H]acetate by incubation with CoA, ATP, Mg2+ and extract from acetone-dried pigeon liver were used. 3. [1−14C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [3H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [3H]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.

show abstract

Section: Extraction Efficiencymentioning

confidence: 83%

mentioning

confidence: 99%

See 1 more Smart Citation

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

Fonnum

1969

Biochemical Journal

1,039

206

View full text Add to dashboard Cite

show abstract

“…In contrast to our study, Kasa (24) detected enzyme activity with copper-lead-thiocholine method in ribosomes, and only in the outer surface of synaptic vesicles and of mitochondria. The controversy about the localization of ChAc in synaptic vesicles between DeRobertis et al (8) and Whittaker et al (43) might be due to species variation of the experimental animals (32). It seems that the presence of ChAc in synaptic vesicles is more acceptable evidence explaining the synthesis and uptake of ACh by vesicles (40).…”

Section: Resultsmentioning

confidence: 92%

Ultracytochemistry of Choline Acetyltransferase in the Neostriatum of the Rat Brain

Kaiya

Iwata

Namba

et al. 1979

Acta Histochem. Cytochem

View full text Add to dashboard Cite

“…On the day of the experiment, a rat was decapitated, the brain quickly removed, weighed, and a 10% homogenate prepared with distilled water. The ChAc assay procedure was a modification by DE BALBIAN VERSTER of the method of SCHRIER and SCHUSTER (1967) and MCCAMAN and HUNT (1965). The final concentration of the buffer substrate was: O-3 M sodium chloride, 0.01 M choline chloride, 2 x lo-4~ physostigmine, 0.02 M magnesium sulphate, 0.05% bovine plasma albumin, 0.08~ potassium phosphate (pH 7.4) and 6.2 x 10m4~ acetyl CoA.…”

Section: Choline Acetyltransferase Assaymentioning

confidence: 99%

Acetylseco hemicholinium-3, a new choline acetyltransferase inhibitor useful in neuropharmacological studies

et al. 1973

View full text Add to dashboard Cite

Summary-Describedare the synthesis and some aspects of the pharmacology of acetylseco hemicholinium-3 (acetylseco HC-3), the acetylated open ring analogue of hemicholinium-3 (HC-3). The effects of both compounds were determined in uiuo on rat brain acetylcholine (ACh), 'Y-choline (14C-Ch) incorporation into 14C-acetylcholine (14C-ACh) and on one way jump box avoidance and escape behavior in naive and trained rats. In addition, the in vitro effects of both drugs were determined on choline acetyltransferase activity (ChAc) in rat brain.When given intraventricularly in doses of l-20 pg both compounds reduced total ACh content in the brain to a maximum of 5'0% of normal in 30-60 min. In doses of 20 pg intraventricularly, both drugs also reduced 14C-Ch incorporation into Y-ACh by 845% for acetylseco HC-3 and by 52% for HC-3.The in viva changes of ACh in the brain were correlated with the behavioral deficits induced in one way shuttle box acquisition and retention. In doses of 20 pg total intraventri~ularly, both compounds produced behavioral deficits which were greater in naive than in trained animals. Zn vitro, acetylseco HC-3 inhibited ChAc activity with an 150 of 1 x 10e5 M with Ch IO-* M and acetyl CoA 6.4 x 10m4 M, while HC-3 had no inhibitory effects. Using rat brain homogenate as the enzyme source and commercial acetyl CoA for kinetic studies, acetylseco HC-3 was shown to be a mixed inhibitor of acetyl CoA and a competitive inhibitor of Ch.The in uiuo actions of acetyiseco HC-3 are consistent with those of a ChAc inhibitor. However, it is necessary to rule out the possibility that the drug may also compete with Ch for its transport across biological membranes like its deacetylated derivative HC-3.The hemicholiniums have been widely investigated since they were first synthesized in 1954 by LONG and SCHUELER. Hemicholinium-3 (HC-3) is the prototype compound of this series. This agent reduces tissue acetyfcholine (ACh), possibly by reducing the active transport of choline (Ch) as shown by MACINTOSH (1963) and HODGKIN and MARTIN (1965). However, other mechanisms such as a shift in Ch metabolism toward phospholipid formation (G~MEz, DOMINO and SELLINGER, 1970a,b;GOMEZ, SELLINGER, SANTIAGO and DOMINO, 1971) and production of a false neurotransmitter through acetyhttion (RODRIGUEZ DE LORES ARNAIZ, LIEBER and DE ROBERTIS, 1970) have been proposed. SCHUELER (1955) found that WC-3 was the most toxic of some 20 bis-quaternary derivatives studied. HC-10 or acetylseco hemicholinium-3 (acetylseco HC-3) produced a toxicological picture similar to HC-3. SCHUELER assumed that acetylseco HC-3 was hydrolyzed in viuo to HC-3. There was little evidence then that acetylseco HC-3 was pharmacologically different from HC-3.

show abstract

Microdetermination of Choline Acetylase in Nervous Tissue

Cited by 364 publications

References 13 publications

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

Ultracytochemistry of Choline Acetyltransferase in the Neostriatum of the Rat Brain

Acetylseco hemicholinium-3, a new choline acetyltransferase inhibitor useful in neuropharmacological studies

Contact Info

Product

Resources

About