Peroxisomal membrane manganese superoxide dismutase: characterization of the isozyme from watermelon (Citrullus lanatus Schrad.) cotyledons.
In this work the manganese superoxide dismutase (Mn-SOD) bound to peroxisomal membranes of watermelon cotyledons (Citrullus lanatus Schrad.) was purified to homogeneity and some of its molecular properties were determined. The stepwise purification procedure consisted of ammonium sulphate fractionation, batch anion-exchange chromatography, and anion-exchange and gel-filtration column chromatography using a fast protein liquid chromatography system. Peroxisomal membrane Mn-SOD (perMn-SOD; EC 1.15.1.1) was purified 5600-fold with a yield of 2.6 mug of enzyme g(-1) of cotyledons, and had a specific activity of 480 U mg(-1) of protein. The native molecular mass determined for perMn-SOD was 108 000 Da, and it was composed of four equal subunits of 27 kDa, which indicates that perMn-SOD is a homotetramer. Ultraviolet and visible absorption spectra of the enzyme showed a shoulder at 275 nm and two absorption maxima at 448 nm and 555 nm, respectively. By isoelectric focusing, a pI of 5.75 was determined for perMn-SOD. In immunoblot assays, purified perMn-SOD was recognized by a polyclonal antibody against Mn-SOD from pea leaves, and the peroxisomal enzyme rapidly dissociated in the presence of dithiothreitol and SDS. The potential binding of the Mn-SOD isozyme to the peroxisomal membrane was confirmed by immunoelectron microscopy analysis. The properties of perMn-SOD and the mitMn-SOD are compared and the possible function in peroxisomal membranes of the peripheral protein Mn-SOD is discussed.
Rodríguez-Serrano
María
M
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080, Granada, Spain.
Romero-Puertas
María C
MC
Pastori
Gabriela M
GM
Corpas
Francisco J
FJ
Sandalio
Luisa M
LM
del Río
Luis A
LA
Palma
José M
JM
D029773:Citrullus / Q000201:enzymology*; D018548:Cotyledon / Q000201:enzymology; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000302:isolation & purification / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D013482:Superoxide Dismutase