Abstract

Pyruvate kinases serve an important role in regulating glycolysis pathways. Different pyruvate kinase isozymes can dictate which of several different products will be formed during the final step of glycolysis. Being able to reliably copy active pyruvate kinases (PK) would allow study of the different isozymes, with the goal of eventually creating plants that are more nutritionally beneficial for humans. This study focused on improving the protocol for replicating each of two isolated PK isozymes from the model plant Arabidopsis thaliana. Previous attempts had resulted in the PK denaturing and consequently precipitating out of solution and so we manipulated the duration of an ice bath prior to lysing the cells in order to encourage chaperone activity, which assists in protein folding. While the several samples under different conditions were produced that contained only our target PK in a soluble form, the sample that had undergone the one hour ice bath contained three times as much of the soluble PK. We successfully produced PK that remained suspended in solution, however we were not able to elicit a measurable oxidation reaction in that solution. This indicates that the PK is inactive, either because it is folded, but not in the correct tertiary structure, or because another factor is needed to allow the PK to catalyze the oxidation reaction. In order to explore what manipulations we can use to selectively encourage the activity of desired pyruvate kinase isozymes we must further explore what additional proteins may be needed to allow active PK to exhibit its activity in a laboratory setting.

Disciplines

Biochemistry | Molecular, Genetic, and Biochemical Nutrition

Mentor

Thomas J Savage

Lab site

California State University, Sacramento (Sac State)

Funding Acknowledgement

This material is based upon work supported by the National Science Foundation under Grant No. 1340110 and is made possible with contributions from the S.D. Bechtel Jr. Foundation, Howard Hughes Medical Institute, Chevron Corporation, National Marine Sanctuary Foundation, and from the host research center. Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the authors. The STAR Program is administered by the Cal Poly Center for Excellence in STEM Education on behalf of the California State University system., This material is based upon work supported by the Howard Hughes Medical Institute and is made possible with contributions from the National Science Foundation under Grant No. 1340110, S.D. Bechtel Jr. Foundation, Chevron Corporation, National Marine Sanctuary Foundation, and from the host research center. Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the authors. The STAR Program is administered by the Cal Poly Center for Excellence in STEM Education on behalf of the California State University system.

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URL: http://digitalcommons.calpoly.edu/star/361

 

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