PMID:6458036

Charette, MF, Henderson, GW and Markovitz, A (1981) ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12.Proc. Natl. Acad. Sci. U.S.A. 78:4728-32

Abstract

Mutations in the lon (capR) gene result in multiple phenotypes, one of which is the failure to degrade abnormal and normal proteins (Deg-). Previous work with partially purified preparations showed that the lon (capR) gene product is a 94,000-dalton polypeptide with an affinity for nucleic acids. The lon (capR) protein has now been highly purified and is demonstrated to have an ATP-dependent protease activity. The enzyme hydrolyzed 3H-labeled alpha-casein into trichloroacetic acid-soluble forms in Tris buffer containing Mg2+ and ATP. The reaction has a pH optimum of 8.5 and ATP was the preferred nucleotide. CTP and UTP could substitute for ATP (75% and 67%, respectively) but GTP, ADP, AMP, cyclic AMP, and PPi could not. Proteolysis by the lon (capR) protein required ATP hydrolysis. Nonhydrolyzable analogs of ATP and CTP did not promote casein cleavage. When low concentrations of ATP were used, proteolysis stopped as the ATP pool was depleted. Casein stimulated lon (capR) ATPase activity, and the products were ADP and inorganic phosphate in equimolar amounts. No protein kinase activity was detected. The DNA-binding activity, present in partially pure preparations, was retained in the purified protein. The gene product purified from a lon nonsense mutant that exhibits the Deg- phenotype (capR9), lacked both the ATP-dependent protease and ATPase activities, though it retained DNA-binding activity. Absence of an ATP-dependent protease activity could account for many of the pleiotropic effects observed in lon mutants.

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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
lon:Gene_Product(s)		GO:0005524	ATP binding	IDA: Inferred from Direct Assay		F	ATPase activity of purified Lon was measured by addition of radiolabeled ATP, either [gamma-32P]ATP or [purine-3H]ATP, and analysis of released Pi by TLC (Figure 7).	complete
lon:Gene_Product(s)		GO:0006508	proteolysis	IDA: Inferred from Direct Assay		P		complete
lon:Gene_Product(s)		GO:0006510	ATP-dependent proteolysis	IDA: Inferred from Direct Assay		P	Purified Lon was assayed for release of radiolabeled Pi from [gamma-32P]ATP and an acid insoluble form of the [3H]-alpha-casein substrate. Release of the products was measured after TCA precipitation by scintillation counting at time points over 4 hours (Figure 6).	complete
lon:Gene_Product(s)		GO:0016887	ATPase activity	IDA: Inferred from Direct Assay		F	Lon is an ATPase, but can also utilize CTP and GTP, though with lower activity.	complete
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