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Title page for ETD etd-07222005-124121


Type of Document Dissertation
Author Clark, Lindsey M.
Author's Email Address lindsey.m.clark@vanderbilt.edu
URN etd-07222005-124121
Title Intracellular Hexokinase Localization in Hybridoma Cultures: Implications for Regulation of Metabolism and Cell Death
Degree PhD
Department Chemical Engineering
Advisory Committee
Advisor Name Title
R. Robert Balcarcel Committee Chair
Fredrick Haselton Committee Member
G. Kane Jennings Committee Member
Kenneth Debelak Committee Member
Robert Tanner Committee Member
Keywords
  • apoptosis
  • mitochondria
  • hexokinase
  • hybridoma
  • batch mammalian cell culture
  • bioprocess
  • glycolysis
Date of Defense 2005-06-22
Availability unrestricted
Abstract

Glycolysis and apoptotic cell death are integrated by the binding of hexokinase to the mitochondria where it has been reported that hexokinase phosphorylates glucose more efficiently and impedes apoptotic protein release into the cytosol by promoting VDAC closure. Interestingly, it has also been reported that the exact binding site for mitochondrial hexokinase is at the VDAC itself, the site where apoptotic proteins are thought to be released from the mitochondria. This study illustrates a phenomenon of decreasing glucose consumption well in advance of apoptotic cell death in batch hybridoma cultures, and an obvious explanation for this decline has yet to be revealed. Given the literature claims that mitochondrial hexokinase exerts regulatory function in terms of glucose phosphorylation rate and apoptotic protein release, then observation of mitochondrial hexokinase detachment in batch cultures would uncover a key molecular phenomenon through which decreasing glucose metabolism and apoptotic protein release may be controlled. Here, a possible occurrence of mitochondrial hexokinase detachment is probed by measuring the in vitro mitochondrial and cytosolic hexokinase activities in samples from batch hybridoma cultures up to 48 hours after subculture. Measurements and statistical treatment of the data reveal that there is little or no change in the distribution of the enzyme between the mitochondrial and cytosolic fractions during batch cultures. However, studies involving chemically-induced mitochondrial hexokinase detachment with clotrimazole show that a re-localization of the enzyme to the cytosol results in a 12-22% decrease in short-term glucose consumption rate for an 8-16 ƒÝM concentration range. Hybridoma cultures receiving 5-20 uM clotrimazole exhibited cell death with apoptotic morphology over a 16-hour exposure period. Furthermore, clotrimazole was observed to induce release of hexokinase from the mitochondria within 1 hour, with levels remaining fairly constant over time after the initial re-localization for concentrations 5 uM or greater. These results are used to discuss the function of mitochondrial hexokinase in regulating glucose metabolism and apoptotic cell death in a biopharmaceutical-producing cell line.

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