PMID: 3748278Jul 1, 1986Paper

Membrane-bound choline acetyltransferase from human brain: purification and properties

Neurochemical Research
J H PengE G McGeer

Abstract

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into a potassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35-65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mumol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChA...Continue Reading

References

Sep 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·H TowbinJ Gordon
Jan 1, 1974·Methods in Enzymology·I ChibataY Matuo
Mar 1, 1972·The Biochemical Journal·D Malthe-Sorenssen, F Fonnum
Jun 18, 1958·The Journal of Physiology·C O HEBB, V P WHITTAKER
May 26, 1962·Nature·E DE ROBERTISA PELLEGRINO DE IRALDI
Jan 1, 1981·Neurochemistry International·J H PengS C Sung

❮ Previous
Next ❯

Citations

Dec 1, 1987·Neuropharmacology·H KobayashiN Matsusaka
Jul 1, 1989·Molecular and Biochemical Parasitology·K H Swamy
Jan 1, 1988·Neurochemistry International·M Docherty, H F Bradford
May 1, 1994·The European Journal of Neuroscience·N SalemL Eder-Colli
Nov 1, 1993·Journal of Neurochemistry·B M Schmidt, R J Rylett
May 1, 1991·Journal of Neurochemistry·M DochertyT H Joh
Dec 1, 1988·Neurochemical Research·R MassarelliJ N Kanfer
Nov 1, 1989·The Canadian Journal of Neurological Sciences. Le Journal Canadien Des Sciences Neurologiques·H F BradfordJ Hardy

❮ Previous
Next ❯

Related Concepts

Related Feeds

Basal Ganglia

Basal Ganglia are a group of subcortical nuclei in the brain associated with control of voluntary motor movements, procedural and habit learning, emotion, and cognition. Here is the latest research.