The functional roles of Ser194, Thr136, Ser140, G1u144, G1u282, and His286 in the subunit of the Na,K-ATPase were studied using site-directed mutagenesis, expression, and kinetic analysis. HeLa cells expressing ouabain-resistant colonies were grown and membrane fractions were prepared. Kinetic characterization of Ser140A1a showed expression levels higher than the control enzyme. The K+K/ values for the activation of the Na,K-ATPase were not altered significantly in all six mutants characterized, although Thr136A1a, Ser140A1a, and His286A1a all displayed a two-fold higher affinity for K. Substitutions for both G1u282A1a and His286A1a showed that these mutants have a two-fold lower affinity for Na than the control enzyme, while Ser94A1a appears to have a five-fold higher affinity for Na. The ATP K/ values were similar to the RD control K/ in all six mutants characterized. In conclusion, all the mutants studied are not cation-coordinating amino acids, but instead affect the Na,K-ATPase conformational transitions required to bind the ions.

• This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

• Malanson, Hunter F.

Publisher

• Worcester Polytechnic Institute

Identifier

• 99D065M

Advisor

• Argüello, José M.

Year

• 1999

Date created

• 1999-01-01

Resource type

• Major Qualifying Project

Major

• Biochemistry

Rights statement

• In Copyright