Date of Award
1993
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyses the biosynthesis of acetylcholine (ACh) from choline and acetylCoenzyme A in cholinergic neurons. Within nerve terminals, this enzyme exists in both cytosolic and membrane-bound forms, although the role of the latter fraction is not clear. ChAT is clearly subject to long-term regulation (over periods of hours to days), likely through alterations in protein synthesis, however very little is known about short-term regulation (seconds to minutes). Therefore, the goals of this study were to determine the mechanisms of short-term regulation of ChAT activity and to elucidate the physiological role of membrane-bound ChAT in ACh biosynthesis.;Using synaptosomes prepared from rat brain hippocampus as a model of intact nerve terminals, approximately 80% of total ChAT activity was found in the cytoplasm and could be further divided into a water-soluble fraction (20%) and sodium phosphate-soluble fraction (60%), while the remaining ChAT (15-20%) appeared to be membrane-bound. Interestingly, only the specific activity of membrane-bound ChAT was significantly increased, in a calcium-dependent manner, following depolarization of intact synaptosomes by both 40 mM KCl and 50 {dollar}\mu{dollar}M veratridine, agents which induce ACh release and subsequent choline uptake. Since W-7, an inhibitor of calcium-calmodulin kinase II, attenuated the depolarization-induced activation of membrane-bound ChAT, protein phosphorylation was examined as a mechanism of short-term regulation. Under resting conditions, only cytosolic ChAT appeared to exist as a phosphoprotein, the abundance of which was calcium-dependent. Furthermore, the pattern of phosphorylation neither correlated with enzyme activity, nor was altered by nerve terminal depolarization. Since the specific activity of cytosolic ChAT was not altered by any experimental manipulation, it was subdivided into water-soluble and sodium phosphate-soluble enzyme fractions. Subsequently, it was found that reduction of intracellular calcium concentration, by lowering the extracellular calcium concentration, reduced membrane-bound ChAT and water-soluble ChAT activities and concomitantly increased sodium phosphate-soluble ChAT activity. These alterations in specific activity were accompanied by parallel changes in both the V{dollar}\sb{lcub}\rm max{rcub}{dollar} and the amount of ChAT-immunoreactive protein in each fraction, suggesting translocation of the enzyme between cytosolic and membrane-bound pools and within cytosolic pools. Finally, to assess the physiological role of membrane-bound ChAT, an experimental strategy was developed based upon the observation that enzyme activity was sensitive to alterations in chloride ion concentration. Under these conditions membrane-bound ChAT did not appear to regulate basal ACh biosynthesis.;In summary, translocation of enzyme protein may be involved in the short-term regulation of ChAT activity within cholinergic nerve terminals. Furthermore, cytosolic ChAT appeared to play the predominant role in the regulation of basal ACh synthesis.
Recommended Citation
Schmidt, Bonnie Marie, "Regulation Of Choline Acetyltransferase In Rat Hippocampal Synaptosomes" (1993). Digitized Theses. 2265.
https://ir.lib.uwo.ca/digitizedtheses/2265