Publication Date

2013

Document Type

Thesis

Committee Members

Norma Adragna (Committee Member), David Cool (Committee Co-chair), Peter Lauf (Advisor)

Degree Name

Master of Science (MS)

Abstract

The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a well-known inhibitor of PKC and cancer growth (Herbert et al., 1990). In human lens epithelial cells (HLECs), CET almost completely inhibits the Na+/K+ pump ATPase (NKA) without changes in phosphorylation (Lauf et al. 2013). Protein alignment studies revealed B-lymphocyte type 2 protein (Bcl-2) BH1- like motifs within the a1 subunit of the NKA to which CET, a drug mimicking Bcl-2 protein BH3-like motifs, might bind thus disrupting NKA function (Lauf et al, 2013). It is unknown by which mechanism CET crosses the plasma membrane to reach its intracellular targets, especially the NKA. In solution, CET exists in two forms, as a positively charged and as uncharged monomer or pseudo-base. The hypothesis is that CET crosses the plasma membrane as monomer and, in the slightly acidic cytosol binds as the positively charged drug to its putative sites on the NKA and pro-survival BcL-2 proteins. The objective of this work is to track the alkaloid CET via MALDI-TOF Mass Spectrometry from the external media bathing HLECs into the cytosol and plasma membrane, if possible into the NKA. A prerequisite of this project is to gain insight into how CET behaves in the biological solutions used, how it might cross the plasma membrane normally impermeable to charged drugs, and how the lower pH within the cytosol affects the ratio of the charged to uncharged species of CET. Once evidence is brought forth that CET reaches the plasma membrane, future studies will address by mass spectrometry, the interactions of CET with the NKA during its canonical operation, and perhaps isolate the CET binding site (s) within the NKA.

Page Count

73

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2013

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