Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization.
A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.
Kalousek
F
F
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
Isaya
G
G
Rosenberg
L E
LE
D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006882:Hydroxyapatites; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D002413:Cations, Divalent; D006882:Hydroxyapatites; D011480:Protease Inhibitors; D011498:Protein Precursors; D012333:RNA, Messenger; D017886:Durapatite; D010450:Endopeptidases; D008666:Metalloendopeptidases; C068038:mitochondrial intermediate peptidase