Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1990

Abstract

Enoyl-(acyl-carrier-protein) reductase was purified from the phytoflagellate Euglena gracilis. Its purification employed DEAE-Sephacel chromatography, Matrex Orange chromatography, and affinity chromatography using acyl carrier protein (ACP) covalently bound to Sepharose as the affinity ligand. Matrex Orange chromatography resolved two different enoyl-ACP reductases having different characteristics. Euglena gracilis appears to resemble higher plants in the possession of two isoforms of this enzyme. Antibodies specific for the cofactor binding site of NADP (H)-requiring dehydrogenases were obtained. They were isolated from a polyclonal population of antibodies directed against yeast glucose-6-phosphate dehydrogenase by affinity chromatography using chicken liver malic enzyme as the affinity ligand. The affinity purified antibodies were covalently bound to Sepharose. Glucose-6-phosphate dehydrogenase and malic enzyme were both bound by the antibody column and were eluted by their cofactor, NADP$\sp+$, identifying the site of recognition of the enzymes by the antibodies as the cofactor binding site. The utility of this antibody affinity column was demonstrated by its ability to bind enoyl-ACP reductase, which was eluted by its cofactor, NADPH. Preliminary studies of the E. gracilis fatty acid synthase (FAS) genes were undertaken using the plasmid pFAS4 (Witkowski et al., 1987), which contains a cDNA insert to part of the rat liver FAS mRNA and was a gift of Dr. Stuart Smith. The insert was cleaved with KpnI and PstI to generate probes specific for the ketoreductase, ACP, and thioesterase domains of the FAS. DNA from wild type E. gracilis and from a mutant, W$\sb{10}$BSML, which lacks chloroplast DNA, was subjected to field inversion gel electrophoresis and the DNA alkaline-blotted onto Nylon membranes. Hybridization of the three probes to the DNA was performed; all three probes hybridized to nuclear DNA, but none of the three hybridized to chloroplast DNA. The three probes also hybridized to a band which was neither nuclear nor cholorplast DNA. This DNA, which was larger than the chloroplast genome, may represent E. gracilis mitochondrial DNA sequences.

Document Type

Dissertation - Open Access

Included in

Biochemistry Commons

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