Title

Alanyl-tRNA Synthetase From Wheat Germ, Purification and cDNA Clone Identification

Date of Award

2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Jeffrey Evans

Advisor Department

Chemistry and Biochemistry

Abstract

Aminoacyl-tRNA synthetases are probably the oldest proteins in protein synthesis machinery. These enzymes prepare the adaptor molecules between the DNA information center and functional manifestation of that information. The adaptor molecules are transfer RNA molecules that are substrates of these enzymes. Alanyl-tRNA synthetase is the enzyme responsible for charging tRNA ala With alanine. This enzyme has been purified and studied in several eukaryotic and prokaryotic organisms. However, no plant alanyl-tRNA synthetase has been purified. We have purified alanyl-tRNA synthetase from wheat germ to near homogeneity. We have isolated several wheat germ cDNA Lambda-gt11 expression clones that cross-reacted with an antibody raised against alanyl-tRNA synthetase from silkworm posterior gland. We have characterized the size and optimum assay conditions of the enzyme and we have measured some of its kinetic parameters. The enzyme appears to be a monomer of approximately 110,000 Kda size. Sequencing of several tryptic fragments of the enzyme revealed several sequences that are conserved in all known alanyl-tRNA predicted protein sequences. Two of the cDNA clones isolated by immunoblotting were partially sequenced. The results were inconclusive as to the nature of the clones. Further investigation of these clones and other isolated clones are necessary to elucidate their nature.