Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree.
An iron-superoxide dismutase (SOD) was purified and characterized from the mature seeds of camphor tree (Cinnamomum camphora). The ultraviolet and visible absorption spectra of camphor Fe-SOD showed patterns typical of cambialistic Fe-SODs. The inductively coupled plasma assay indicated that there was 0.5-1 atom of Fe(2+) per camphor Fe-SOD subunit. The cDNA of camphor Fe-SOD, including the coding region and the 3' noncoding region, was obtained by reverse transcription polymerase chain reaction using the total RNA from immature seeds of C. camphora as template and then sequenced. The complete amino acid sequence of camphor Fe-SOD was deduced from the cDNA sequence. The correctness of the amino acid sequence was confirmed by directly sequencing five peptide fragments of the enzyme. The molecular mass calculated for the camphor Fe-SOD subunit from its 204 amino acid residues was 22,930.6 Da, The cDNA of camphor Fe-SOD was cloned into the expression vector PMFT7-5 and then expressed in Escherichia coli strain BL21. The reconstructed Fe- or Mn-SOD was purified to homogeneity through column chromatography. Activity of the Fe- or Mn-SOD was found to be almost equal to that of natural camphor Fe-SOD, which is the first cambialistic SOD isolated from eukaryotic cells.
Chen
Huai-Yang
HY
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Hu
Rong-Gui
RG
Wang
Bao-Zhong
BZ
Chen
Wen-Feng
WF
Liu
Wang-Yi
WY
Schröder
Werner
W
Frank
Peter
P
Ulbrich
Norbert
N
D001483:Base Sequence; D027423:Cinnamomum camphora / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000737:chemistry / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D012639:Seeds / Q000737:chemistry / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase