Purification of catalase from pea leaf peroxisomes: identification of five different isoforms.
Catalase activity was analyzed in seven organs of pea (Pisum sativum L.) plants: leaves, seeds, flowers, shoots, whole fruits, pods and roots. Leaves showed the highest activity followed by whole fruits and flowers. Catalase was purified from pea leaf peroxisomes. These organelles were isolated from leaves by differential and sucrose density-gradient centrifugation, and catalase was purified by two steps involving anion exchange and hydrophobic chromatography using a Fast Protein Liquid Chromatography system. Pure catalase had a specific activity of 953 mmol H2O2 min(-1) mg(-1) protein and was purified 1000-fold, with a yield of about 19 microg enzyme per kg of pea leaves. Analysis by SDS-PAGE and immunoblot showed that the pea catalase was composed of subunits of 57 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 252 and 400 nm with molar extinction coefficients of 2.14 x 10(6) and 7.56 x 10(6) M(-1) cm(-1), respectively. By isoelectric focusing (pH 5-7), five different isoforms were identified and designated as CAT1-5, with isoelectric points of 6.41, 6.36, 6.16, 6.13 and 6.09, respectively. All the catalase isoforms contained a subunit of 57 kDa. Post-embedment, EM immunogold labelling of catalase showed a uniform distribution of the enzyme inside the matrix and core of pea leaf peroxisomes.
Corpas
F J
FJ
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Granada, Spain. javier.corpas@eez.csic.es
Palma
J M
JM
Sandalio
L M
LM
López-Huertas
E
E
Romero-Puertas
M C
MC
Barroso
J B
JB
Del Río
L A
LA
D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology* / Q000648:ultrastructure; D020675:Peroxisomes / Q000201:enzymology / Q000648:ultrastructure; D018515:Plant Leaves / Q000201:enzymology / Q000648:ultrastructure; D020836:Protein Structure, Quaternary; D013053:Spectrophotometry; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution
D007527:Isoenzymes; D002374:Catalase