| Summary: | The purification of citrate synthase from E. coli (wild-type) is described. The subunit Mr value was determined to be approximately 47,000 by SDS-polyacrylamide gel electrophoresis. The hexameric nature of this enzyme was established using bifunctional cross-linking reagents and analysis by SDS-polacrylamide gel electrophoresis. The purification of citrate synthase from a strain of E. coli containing elevated levels of the enzyme is also described. This organism possesses the qlt A gene on several copies of a hybrid plasmid and the structural and regulatory properties of its citrate synthase were found to be identical to the wild-type E. coli enzyme in all aspects studied. The implications of producing elevated levels of wild-type and mutant citrate synthase are discussed. Purification of citrate synthase from B. megaterium is reported. The enzyme was found to have a native Mr of 84,000 by a combination of analytical ultracentrifugation and gel filtration. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions revealed a subunit of Mr value 39,000 - 43,000. The enzyme was discovered to be dimeric by the use of cross-linking reagents and analysis by SDS-polyacrylamide gel electrophoresis, Modification of B. megaterium citrate synthase by chemical reagents was performed. The enzyme was insensitive to the thiol-specific reagent DTNB but inactivated by DEPC which specifically attacks histidine moieties. Spectroscopic analysis of the inactivated enzyme revealed 2 histidines modified per dimer necessary for a 100% loss in catalytic activity. Protection against inactivation by DEPC was afforded by both substrates and ATP. The purification of citrate synthase from a mutant strain of E. coli is described. The enzyme was found to have a native Mr value of 76,000 and a subunit Mr 42,000 - 43,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively. Comparisons of the structural and functional properties of citrate synthases from various sources are discussed in the light of the sum total of these findings.
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