EVIDENCE FOR A NOVEL ATP-DEPENDENT PROTEASE FROM THE RAT-LIVER MITOCHONDRIAL INTERMEMBRANE SPACE - PURIFICATION AND CHARACTERIZATION

An ATP-dependent protease in the intermembrane space of rat liver mito chondria, MISP I (mitochondrial intermembrane space protease), was par tially purified and characterised. The protease complex has a molecula r mass of 200 kDa and appears to be an oligomeric enzyme complex. The proteolytic activity of the enzyme can be stimulated up to S-fold by M g(2+)ATP. The K-m for ATP is 200 mu M. Nucleoside triphosphates, but n ot ADP, AMP, or nonhydrolysable ATP analogues, can substitute for ATP, The protease exhibits multicatalytic properties with chymotrypsin-lik e, peptidyl-glutamyl-hydrolysing, and trypsinlike activities, Of the l atter the trypsin-like activity is not enhanced by ATP. In addition to the hydrolysis of fluorogenic peptide substrates the protease is able to degrade radiolabeled model proteins. The ATP-dependent mitochondri al protease was characterised as a cysteine protease sensitive to hemi ne, The cross reactivity of an anti-human-S4 antibody raised against a n ATPase subunit of the PA700 complex with a component of MISP I indic ated a structural relationship, Furthermore, ATP-agarose-binding assay s revealed the connection of the peptide hydrolysing activity with an ATP binding domain, The data presented here and a comparison with know n ATP-dependent mitochondrial proteases demonstrated that MISP I repre sents a novel ATP-dependent protease in the mitochondrial intermembran e space of rat liver.