Date of Award
11-5-2002
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
First Advisor
Katherine A Sukalski
Abstract
Interactions between the glycolytic enzymes lactate dehydrogenase A (LDHA), lactate dehydrogenase B (LDHB) and aldolase and F-actin o f the cytoskeleton were studied by constructing enzyme/GFP (green fluorescent protein) fusion proteins and measuring the interactions via in vitro co-precipitation analysis and in vivo localization analysis using fluorescent and confocal microscopy. Primers were used to isolate the genes o f LDHA, LDHB and aldolase from human muscle and heart cDNA libraries by PCR. GFP-fusion proteins were designed in N-terminal and C-terminal directions. GFPfusion proteins containing the 10-terminal amino acids from both the N and C terminus o f these enzymes were also prepared. Bacterial fusions that were used in co-precipitation experiments interacted with mammalian F-actin with the same affinity as GFP except for C-terminal GFP-aldolase, which expressed 3X the binding o f the other constructs. Eukaryotic fusions were expressed in 3T3 cells and localization o f LDHA, LDHB, aldolase and the peptide fusions within these cells, determined by microscopy. GFP and the constructed fusion proteins appeared to associate with F-actin along stress fibers within these cells.
Recommended Citation
Wald, Karl Alexander, "Protein -protein interactions between aldolase, lactate dehydrogenase and the cytoskeleton using green fluorescent protein-labeled enzymes." (2002). Theses and Dissertations. 7854.
https://commons.und.edu/theses/7854