Synthesis and processing of the precursor for human mangano-superoxide dismutase.
Superoxide dismutase comprises a family of metalloenzymes that catalyze the oxido-reduction of superoxide anion to H2O2. Manganese superoxide dismutase (Mn-SOD) is encoded by nuclear chromatin, synthesized in the cytosol, and imported posttranslationally into the mitochondrial matrix. We isolated and sequenced complementary DNA encoding human Mn-SOD. The Mn-SOD cDNA was 1001 base pairs long with a single open reading frame. It contained 95 base pairs of 5' untranslated sequence, and 216 base pairs of 3' untranslated sequence, followed by a short polyadenylation tract. The deduced amino acid sequence suggests a mature protein of 198 amino acids preceded by a 24 amino acid leader peptide. A major transcript of 1000 nucleotides was identified by hybridization of the cDNA with RNA isolated from human cells. Precursor Mn-SOD was produced by in vitro transcription of the human Mn-SOD cDNA followed by in vitro translation utilizing rabbit reticulocyte lysate. The primary translation product of the cDNA is a polypeptide of Mr 26,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. When the Mr 26,000 propeptide was incubated with freshly isolated rat liver mitochondria, the peptide was proteolytically processed to a Mr 24,000 polypeptide. Proteolytic processing was accompanied by an energy-dependent import of the peptide into the isolated liver mitochondria. Mature 125I-labelled Mn-SOD, isolated from rabbit liver, was not imported in vitro into mitochondria, indicating that the energy-dependent uptake of Mn-SOD by liver mitochondria was specific for the Mn-SOD precursor.
Wispé
J R
JR
Department of Pediatrics, University of Cincinnati, OH 45267-0541.
Clark
J C
JC
Burhans
M S
MS
Kropp
K E
KE
Korfhagen
T R
TR
Whitsett
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000032:analysis; D004247:DNA / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms / Q000032:analysis; D008168:Lung / Q000032:analysis; D008345:Manganese; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics
D011498:Protein Precursors; D012333:RNA, Messenger; D008345:Manganese; D012313:RNA; D004247:DNA; D013482:Superoxide Dismutase