Semester

Fall

Date of Graduation

2002

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Biochemistry

Committee Chair

Jim Mahaney.

Abstract

The Ca-ATPase of sarcoplasmic reticulum removes cytosolic calcium to promote muscle relaxation. In the heart, the Ca-ATPase is regulated by phospholamban, which inhibits the Ca-ATPase by decreasing Ca-ATPase calcium sensitivity. However, the kinetic and thermodynamic mechanisms of inhibition are not understood. The purpose of this research was to test the hypothesis that phospholamban regulates Ca-ATPase kinetics by increasing Ca-ATPase activation energy. The baculovirus-insect cell expression system was used to produce samples containing Ca-ATPase alone or Ca-ATPase with phospholamban. The temperature-dependence of Ca-ATPase activity and catalytic site density was measured in the absence and presence of phospholamban at sub-saturating calcium and used to calculate the temperature-dependence of Ca-ATPase turnover. Arrhenius analyses showed that phospholamban increased Ca-ATPase activation energy from 31 +/- 3 J/mol (Ca-ATPase only) to 52 +/- 5 J/mol (Ca-ATPase + phospholamban). The results supported the hypothesis and provided new insight into the mechanism of phospholamban inhibition of Ca-ATPase.

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