Date of Award

3-19-1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Harvey Knull

Abstract

Interactions between glycolytic enzymes and cytoskeletal proteins are electrostatic in nature. Studies conducted with derivatizing agents show that the amino side chains of basic amino acids on aldolase interact with carboxyl side chains of acidic amino acids on tubulin. In this study we used the techniques of covalent cross-linking, non-enzymatic cleavage and enzymatic cleavage to determine the amino acids in actin which are involved in the actin-aldolase interaction.Actin and aldolase were covalently cross-linked using the zero length cross-linker 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide to form a direct covalent link between the two proteins. The cross-linking procedure resulted in the formation of one major product with a molecular weight of approximately 80 kilodaltons as determined by gel electrophoresis, consistent with a single cross-linking site between monomers of the proteins. The cross-linked product contained both actin and aldolase as indicated by banding patterns consistent with the parental proteins upon Cleveland digestion. The presence of unique bands in the digest was presumably due to cross-linked peptides.Digestion of the cross-linked product with hydroxylamine yielded three cross-linked peptides with molecular weights of 41, 55 and 62 kilodaltons. The predominant 41 kilodalton band ran equidistant between native actin and native aldolase.C-terminal sequencing of the 41 kilodalton band was performed with carboxypeptidase Y which allowed for the selective cleavage of the peptide bonds involving neutral or acidic amino acids. Released amino acids, derivatized with dabsyl chloride and identified by high performance liquid chromatography included residues of the amino terminal fragment of actin in addition to the C-terminal amino acids of both actin and aldolase. The digest resulted in the release of only one aspartate and one glutamate which indicates that aspartate-3 is involved in the bond in the 41 kilodalton cross-linked peptide. Molecular weight analysis of the 55 kilodalton cross-linked peptide is consistent with a binding site involving the N-terminal fragment of actin.

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