Masters Thesis

Identifying acetylcholinesterase oligomerization states in Tritonia diomedea tissues

Tritonia diomedea (also known as Tritonia tetraquetra) is a marine gastropod that is used to study how the neuronal system controls behavior. Acetylcholinesterase (AChE) plays an essential role in neurotransmission as it terminates cholinergic signaling in many species. The different oligomerization states of AChE were analyzed in T. diomedea’s hemolymph and central nervous system (CNS), consisting of brain and buccal ganglia for which fully soluble and detergent soluble extracts were obtained. Blue native polyacrylamide gel electrophoresis (BN-PAGE) gels were stained for AChE activity and showed different staining patterns for hemolymph with four bands and CNS tissues with two bands. Each band represents a different oligomerization state. Sedimentation profiles obtained via sucrose density gradient ultracentrifugation also demonstrated that AChE occurs in different oligomerization states in the hemolymph compared to CNS tissues. The two most prominent forms of hemolymph AChE had sedimentation coefficients of 3.0 ± 0.8 S and 12.0 ± 1.6 S. For all CNS tissues the large peak around 12 S was absent and the main peak in the buccal ganglia samples had sedimentation coefficients that were smaller than 3 S. Only the FS brain sample appeared to have a peak around 4.9 S. The hemolymph, brain FS extract, and buccal ganglia extracts, however, shared a BN-PAGE band that exceeded the position of the largest molecular weight marker (>1236 kDa). This large molecular form was also detected in sucrose density gradient ultracentrifugation where a tailing peak was observed at the end of the sedimentation profile. This large molecular form could potentially be an aggregation or precipitation since it did not appear in tissue extracts containing detergent. In sum, the CNS tissues of Tritonia diomedea contained fully soluble and detergent soluble forms of AChE. Hemolymph AChE was in a fully soluble form. With the exception of a potentially large molecular form, the AChE oligomerization states detected via BN-PAGE and sucrose density gradient ultracentrifugation differ between the hemolymph and the CNS tissue.