Solution kinetics analysis of vesicle trafficking of zymogen granule.

Abstract

Zymogen granule (ZG) is a micrometer size enzymatic vesicle in the exocrine pancreas. It regulates secretion of digestive enzymes. It is also associated with pancreatic disease and disorder. It has several myosin classes molecular motors such as 1c, 5c, 6 and 7b. These myosin motors are responsible for transportation of ZG. The hydrolysis of adenosine triphosphate (ATP) initiates the directional motion of these myosin motors. To understand what the key part of the kinetics step for the ZG's trafficking, we measured each step of ATPase of purified ZGs from the rat pancreas. In the present study, we employed the stop flow techniques in order to investigate step(s) of ATPase cycle, e.g. ATP binding, ADP dissociation, phosphate release, and actin binding rate. The data were observed as ensemble work of myosins. The results provide the kinetics of these molecular motors complex present in the ZG. For ATP binding, mant-dATP was used, and result was fit with a single exponential with about 200 (/s) rate. For ADP dissociation, mant-dADP was used with double mixing methods. The fast and slow rates of ADP dissociation are 120 /s and 28 /s, respectively. For actin binding rate, Pyrene-actin was used. The fitting curves were found to be double exponential rates. The fast and slow rates are 64 /s and 12 /s, respectively. For the phosphate release, Phosphate binding protein was used and was observed to be double exponential, and the fast and slow rates are 250 /s and 8/s, respectively. Comparing only Myosin 5c, slow rate part of ADP dissociation and phosphate dissociation shows a little more significance, while ATP binding rate is not different.