Off-campus WSU users: To download campus access dissertations, please use the following link to log into our proxy server with your WSU access ID and password, then click the "Off-campus Download" button below.
Non-WSU users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Access Type
WSU Access
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Physics and Astronomy
First Advisor
TAKESHI SAKAMOTO
Abstract
Zymogen granules (ZGs) are about 1 μm diameter enzymatic vesicles found in the exocrine pancreas and act as inactive precursors of digestive enzymes. Gel electrophoresis and mass spectroscopy had shown presence of myosins motors like myosin 1c, 5c, 6 and 7b at the surface of the ZGs and contribute to ZGs transportation in cells. Myosin motors utilize ATP as an energy to function in the actin filaments. Among different classes of myosins, some motors involve in the vesicle trafficking. Vesicle trafficking motors have special features such as they can move in actin tracks continuously and have high duty ratio. For e.g. myosin 5a transports neuronal vesicles (diameter: 40 nm) and has high duty ratio. The high duty ratio motors bound to actin more than half of the ATPase cycle. In contrast, myosin 1c and myosin 5c are low duty ratio motors and cannot move in the actin filament continuously. From the previous study of two myosin 5c molecules using DNA origami in our lab had shown continuous move in the actin filament and demonstrated the possibility of two myosin 5c to transport ZGs which also have flatter surface curvature than neuronal vesicle. In this study, we measured the key steps of ATPase cycle of purified ZGs for the first time from rat pancreas. The duty ratio for ZG was found to be more than that of a single molecule of two headed myosin 5c. The high duty ratio was contributed by phosphate release and low value of mantADP dissociation.108 Myosin16A (Myo16A) is single headed motor. It has potential function during early neonatal brain development. One of the interesting features of aves and mammalian Myo16A is the presence of cysteine (C) residue at the switch 1 of the ATP binding pocket instead of arginine (R). This residue has demonstrated evolutionary change in the vertebrate development. Our work demonstrated the key rates of actin activated. ATPase cycle for the Myo16A-C552 and substitution Myo16A-C552R were studied. The maximum ATPase and KM of actin were measured as 0.52 s-1 and 2.0 μM for Myo16A-C552R, indicating that Myo16A- C552R is a slow motor and weakly binding to actin filament. Myo16A-C552 showed slower rates for actin binding, mantATP binding, and phosphate release. However, mantADP release showed double exponential such as 180 s-1 fast (~73%) and 22 s-1 (~27%) slow rates for WT and 243 s-1 fast (85%) and 17 s-1 slow (15%) rates for substitution Myo16A-C552R. The substitution C552R had slowed the phosphate release rates by 9 times. This result signified the role of arginine at the ATP binding pocket. The duty ratio of Myo16A-C552 was observed to be < 23% and was lower than high duty ratio myosin 5a (70%) motor.
Recommended Citation
Pangeni, Susheel, "Solution Kinetic Study Of Vesicle Trafficking Of Zymogen Granules And Myosin 16a Molecular Motor" (2024). Wayne State University Dissertations. 4105.
https://digitalcommons.wayne.edu/oa_dissertations/4105