AbstractTwo distinct citrate synthases were found in a mutant Pseudomonas aeruginosa. A 'large' enzyme (CS I; mol.wt.=300,000), allosterically inhibited by NADH and activated by AMP, predominates in a log-phase (nutrient- glutamate) culture whereas a 'small' (non-regulated) enzyme (CS II; mol.wt.=100,000) predominates in stationary phase. By contrast, wild-type Ps.aeruginosa contains only one citrate synthase - a 'large' enzyme, allosterically inhibited by NADH and deinhibited by AMP. This growth- stage dependent enzyme variation in the mutant represents a quite novel finding. Growth on different carbon sources revealed considerable variation in the ratio of the two mutant enzymes. It appears that CS II is not a breakdown product of CS I. Treatment of a growing nutrient-glutamate culture with chloramphenicol resulted in no change in the ratio of the two enzymes during stationary phase; indicating the probability of de novo synthesis of CS II. Matrex Gel Red A, a group-specific ligand, was used in the purification of the Pseudomonas citrate synthases and, together with other conventional procedures, resulted in considerable purification of all three enzymes. Several affinity ligands were prepared using substrates and effectors of citrate synthase but none was useful in the purification of the Pseudomonas enzymes. The catalytic and regulatory properties of the wild-type and mutant enzymes were compared. Photooxidation with Methylene Blue and treatment with the thiol-blocking reagent 5,5'-dithiobis-(2-nitrobenzoic acid) both resulted in a rapid loss of sensitivity to inhibition by NADH and a slower loss of activation by AMP, indicating that the enzyme contains separate NADH and AMP binding sites. A similar situation may occur in the wild-type enzyme. Considerable variation was found in both the sensitivity of diverse citrate synthases to various thiol-blocking reagents and the effect of salt on the reactivity of these reagents.
|Date of Award||1981|
Studies on Pseudomonas aeruginosa citrate synthase.
Solomon, M. (Author). 1981
Student thesis: Doctoral Thesis › PhD