This study presented evidence for the existence of a membrane-bound, sodium-activated ATPase which does not require potassium ions for its activity. Furthermore, the data suggest the presence of a soluble factor which activates this enzyme and may play a regulatory role in sodium transport.

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡ÃƒÂªE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xi, 82 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / The multi-protein complex of F0F1 ATP synthase has been of great interest in the fields of microbiology and biochemistry, due to the ubiquitous use of ATP as a biological energy source. Efforts to better understand this complex have been made through structural determination of segments based on NMR and crystallographic data. Some experiments have provided useful data, while others have brought up more questions, especially when structures and functions are compared between bacteria and species with chloroplasts or mitochondria. The epsilon subunit is thought to play a signi cant role in the regulation of ATP synthesis and hydrolysis, yet the exact pathway is unknown due to the experimental difficulty in obtaining data along the transition pathway. Given starting and end point protein crystal structures, the transition pathway of the epsilon subunit was examined through computer simulation.The purpose of this investigation is to determine the likelihood of one such proposed mechanism for the involvement of the epsilon subunit in ATP regulation in bacterial species such as E. coli.

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus affects an estimated 285 million people worldwide, and a central component of diabetes pathophysiology is diminished pancreatic islet beta cell function resulting in the inability to manage blood glucose effectively. The beta cell is a highly specialized metabolic factory that possesses a number of specialized characteristics, chief among these a highly developed endoplasmic reticulum (ER). The sarco endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen, and while the Pancreatic and duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in beta cell development and function, recent data also implicate Pdx-1 in the maintenance of ER health. Our data demonstrates that a decrease of beta cell Pdx-1 occurs in parallel with decreased SERCA2b expression in models of diabetes, while in silico analysis of the SERCA2b promoter reveals multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. Results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using an ER-targeted D4ER adenovirus and fluorescence lifetime imaging microscopy. Co-transfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity three-fold relative to the empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/- mice were fed high fat diet for 8 weeks. Isolated islets from these mice demonstrated increased expression of spliced Xbp1, signifying ER stress, while subsequent SERCA2b overexpression in isolated islets reduced spliced Xbp1 levels to that of wild-type controls. These results identify SERCA2b as a direct transcriptional target of Pdx-1 and define a novel role for altered ER Ca2+ regulation in Pdx-1 deficient states.