Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1993

Abstract

Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a substrate. Comparisons with other ACPs included the following items: biological activity, pI, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule. ACPs from E. coli and Euglena have been shown to interact with melittin, a cationic peptide from bee venom. E. coli ACP is a small (Mr, 8847), acidic, Ca$\sp{++}$-binding protein which possesses some characteristics resembling those of regulatory Ca$\sp{++}$-binding proteins including interaction with melittin. Melittin inhibited activity of the nonaggregated FAS from Euglena using either E. coli or Euglena ACP as a substrate. The peptide also inhibited activity of the aggregated FAS from Euglena. Antibodies against melittin were raised. Anti-melittin inhibited activity of both the nonaggregated and aggregated FAS enzyme systems from Euglena relative to nonimmune antibody. Investigation of inhibition of the nonaggregated FAS enzyme system demonstrated that acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and keto-acyl-ACP synthetase activities were inhibited to different degrees by anti-melittin antibodies, while keto-acyl-ACP reductase and enoyl-ACP reductase enzyme activities were not inhibited.

Document Type

Dissertation - Open Access

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