Title page for ETD etd-08182010-170845

Type of Document Dissertation
Author Sivakumaran, Vidhya
URN etd-08182010-170845
Title Physical Mechanisms of Ca-ATPase Regulation in the Heart
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Mahaney, James E. Committee Chair
Dolan, Erin L. Committee Member
Kennelly, Peter J. Committee Member
Wyeth, Richard P. Committee Member
  • SERCA2a
  • Ca-ATPase
  • Phospholamban
  • EPR
  • Fluorescence
Date of Defense 2010-08-09
Availability unrestricted
The Ca-ATPase is an integral membrane enzyme which translocates two calcium ions from the cytoplasm of the cell to the sarcoplasmic reticulum lumen utilizing ATP breakdown as its energy source, in order to promote muscle relaxation. The focus of this research is the cardiac isoform of the Ca-ATPase which undergoes allosteric regulation by the phosphoprotein phospholamban (PLN). The Ca-ATPase is thought to be a target for nitrative stress and is affected by several chronic diseases of the heart.

In the heart, age-based nitration of the Ca-ATPase inhibits Ca2+ transport activity but the physical mechanism by which nitration inhibits Ca-ATPase activity is not understood. Conversely, nitroxyl (HNO), a new candidate for drug therapy for congestive heart failure (CHF), improves overall cardiovascular function by increasing Ca-ATPase activity in the heart. However, the physical mechanism for this activation is unknown. Therefore, we have used enzyme kinetics, fluorescence spectroscopy, and EPR spectroscopy studies to determine the effects of ONOO- and HNO on the Ca-ATPase and the physical regulation of the Ca-ATPase by PLN.

Treatment of Ca-ATPase with a nitrating agent, ONOO-, inhibited Ca-ATPase activity, and the [ONOO-]-dependent inhibition of the Ca-ATPase was more effective in the presence of PLN. ONOO- did not affect the [Ca2+]-dependence of Ca-ATPase activity either in the presence or absence of PLN. ONOO- had no effect on Ca-ATPase rotational mobility or oligomeric interactions, as affected by PLN, but ONOO- decreased the amplitude of the Ca2+-dependent E2 to E1•Ca2 conformational change, both in the absence and presence of PLN.

Treatment with HNO had no affect on the [Ca2+]-dependence of Ca-ATPase activity in the absence of PLN; however in the presence of PLN, the [Ca2+]-dependent activity was shifted to lower Ca2+ levels and corresponded to the uncoupling of PLN from the Ca-ATPase. HNO decreased Ca-ATPase rotational mobility and increased the Ca-ATPase Ca2+-dependent conformational transition, consistent with uncoupling PLN from the Ca-ATPase.

Taken together, these results suggest that ONOO- inactivates a fraction of enzyme units to lower overall enzyme activity, whereas HNO uncouples PLN from the Ca-ATPase with increases in Ca-ATPase conformational flexibility and Ca-ATPase activity.

  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  Sivakumaran_V_D_2010.pdf 2.52 Mb 00:11:40 00:06:00 00:05:15 00:02:37 00:00:13
  Sivakumaran_V_D_2010_copyright.pdf 7.60 Mb 00:35:10 00:18:05 00:15:49 00:07:54 00:00:40

Browse All Available ETDs by ( Author | Department )

dla home
etds imagebase journals news ereserve special collections
virgnia tech home contact dla university libraries

If you have questions or technical problems, please Contact DLA.