Isolation and purification of mitochondrial Mn-superoxide dismutase from the gymnosperm Pinus sylvestris L.
Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1) was purified from germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition. The purification schedule included (NH4)2SO4 fractionation, anion-exchange and hydrophobic-interaction chromatographies and chromatofocusing. Purified Mn-SOD had an apparent specific activity of 4,130 McCord-Fridovich units (mg protein)-1. The molecular mass of the holoenzyme was estimated to be 91 kDa by size-exclusion chromatography, and a molecular mass of 23 kDa was determined by SDS-PAGE. However, isoelectric focusing demonstrated that the purified enzyme consisted of three similarly migrating isoforms, with isoelectric points of approximately 6.5. NH2-terminal amino acid sequencing of purified Mn-SOD revealed no differences among the three isoforms. The comparison of the first 32 NH2-terminal amino acids with sequences of NH2-terminal amino acids of Mn-SODs from angiosperms reflected the phylogenetic distances between Scots pine, which is a gymnosperm, and angiospermic species. Cell fractionation suggested the mitochondrial localization of Mn-SODs and no evidence for glyoxysomal localization was found. Mn-SOD activity was absent from dry seeds. It was detectable at a considerable level after imbibition for 24 h, and it was again absent from 3-week-old seedlings.
Streller
S
S
Department of Forest Genetics and Plant Physiology, Faculty of Forestry, Swedish University of Agricultural Sciences, Umeå.
Krömer
S
S
Wingsle
G
G
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D041605:Pinus sylvestris; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000201:enzymology; D013482:Superoxide Dismutase / Q000302:isolation & purification*
D013482:Superoxide Dismutase