PURIFICATION AND CHARACTERIZATION OF A SUBSTRATE PROTEIN FOR MITOCHONDRIAL ATP-DEPENDENT PROTEASE IN BOVINE ADRENAL-CORTEX

We have purified SP-22, a substrate protein for mitochondrial ATP-depe ndent protease in bovine adrenal cortex. Native SP-22 showed an M(r) o f 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation o f adrenocortical tissues revealed that SP-22 was localized in the mito chondrial matrix, suggesting that SP-22 is a natural substrate for ATP -dependent protease, a matrix enzyme. The concentration of SP-22 in ad renocortical mitochondrial fractions was 16 +/- 3 mu g/mg proteins (me an +/- SD, n=6) as determined by radioimmunoassay using specific anti- SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid seque nce of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fr agment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% h omologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in S almonella typhimurium, thiol-specific antioxidant in Saccharomyces cer evisiae, and some other proteins. Since a segment around AA47 was high ly conserved, this residue may be important for the biochemical functi ons of SP-22.