Evidence for a novel ATP-dependent protease from the rat liver mitochondrial intermembrane space: purification and characterisation.
An ATP-dependent protease in the intermembrane space of rat liver mitochondria, MISP I (mitochondrial intermembrane space protease), was partially purified and characterised. The protease complex has a molecular mass of 200 kDa and appears to be an oligomeric enzyme complex. The proteolytic activity of the enzyme can be stimulated up to 3-fold by Mg2+ATP. The Km for ATP is 200 microM. Nucleoside triphosphates, but not ADP, AMP, or nonhydrolysable ATP analogues, can substitute for ATP. The protease exhibits multicatalytic properties with chymotrypsin-like, peptidyl-glutamyl-hydrolysing, and trypsin-like activities. Of the latter 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 mitochondrial protease was characterised as a cysteine protease sensitive to hemine. The cross reactivity of an anti-human-S4 antibody raised against an ATPase subunit of the PA700 complex with a component of MISP I indicated a structural relationship. Furthermore, ATP-agarose-binding assays revealed the connection of the peptide hydrolysing activity with an ATP binding domain. The data presented here and a comparison with known ATP-dependent mitochondrial proteases demonstrated that MISP I represents a novel ATP-dependent protease in the mitochondrial intermembrane space of rat liver.
Sitte
N
N
Institute of Biochemistry, Medical Faculty (Charité), Humboldt University Berlin, Hessische Str. 3-4, D-10115 Berlin, Germany. nsitte@rz.charite.hu-berlin.de
Dubiel
W
W
Kloetzel
P M
PM
D049069:ATP-Dependent Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D010450:Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000737:chemistry / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*
D009711:Nucleotides; D011480:Protease Inhibitors; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C111985:mitochondrial intermembrane space protease