Studies of the ATP-dependent proteolytic enzyme, protease La, from Escherichia coli.

The ATP-dependent proteolytic activity, protease La, *om Escherichia coli has been partially purified, and the role of ATP investigated.ATP (3 m) stimulated degradation of [methyL3HJcasein and [methyl-'*C]apohemoglobin 8-40-fold but only when M&' was present.The nucleotide had to be continuously present for this process, since rapid depletion of ATP prevented further proteolysis.A concentration of 260 BM MgATP gave half-maximal activity, but the effects of different concentrations of ATP depended also on the Mg' concentration, and high concentrations of nucleotides inhibited.dATP, CTP, and U T P also stimulated proteolysis but not as well as ATP, while GTP, ADP, AMP, pyrophosphate, and the nonmetabolizable analogs a,/3-methylene-ATP, B,y-methylene-ATP, adenosine 5'-0-(3-thiotriphosphate), and 2',3'-dialdehyde-ATP, had little or no effect.These analogs, as well as ADP and AMP, inhibited the effect of ATP, probably by competition for the same binding sites.Both ATP and ADP appeared to bind to and stabilize the protease (even in the absence of M&+), since they prevented the rapid loss of activity that occurred at 42 "C.No protein kinase or protein adenylylase activity was demonstrable in these preparations.ATP-dependent proteolysis also was not affected by the addition of termination factor riw or ubiquitin, and these preparations did not conjugate ubiquitin to protein substrates.Although certain inhibitors of ATPases (NJV" dicyclohexylcarbodiimide, oligomycin, azide) did not affect casein hydrolysis, others (vanadate, Dio-9, quercetin) markedly inhibited this process.Proteolysis ceased rapidly after addition of vanadate; thus, continued splitting of ATP seems necessary, but a simple relationship between ATPase activity and proteolysis was not clear in these impure preparations.At concentrations above 200 m, phosphate markedly inhibited the ATP-dependent activity and stimulated proteolysis slightly in the absence of ATP.These various properties suggest that ATP cleavage is required for proteolysis and distinguish protease La from the recA gene product and the cytoplasmic ATPstimulated proteases of mammalian cells.One of the fundamental and unexplained features about intracellular protein degradation is its apparent requirement