Characterization of endoproteases from plant peroxisomes.
In this work, the characterization of endoprotease (EP) isoenzymes in peroxisomes is reported for the first time in cell organelles purified from pea leaves (Pisum sativum L.). A comparative analysis of the endo-proteolytic activity in peroxisomes purified from young (15-day-old) and senescent (50-day-old) leaves was carried out. Peroxisomes purified from senescent leaves showed a much higher endo-proteolytic activity than organelles from young plants. A 16 h incubation with exogenous substrates was the threshold time for the detection of a linear increase in the endo-proteolytic activity of peroxisomes from senescent leaves. Three EP isoenzymes (EP2, EP4 and EP5), having molecular masses of 88, 64 and 50 kDa respectively, were found in young plants by using SDS/polyacrylamide-gradient gels co-polymerized with gelatin. However, four additional isoenzymes (EP1, EP3, EP6 and EP7), with molecular masses of 220, 76, 46 and 34 kDa respectively, were detected in senescent plants. All the isoenzymes detected in peroxisomes from both young and senescent leaves were neutral proteases. By using different class-specific inhibitors, the electrophoretically separated EP isoenzymes were characterized as three serine-proteinases (EP1, EP3 and EP4), two cysteine-proteinases (EP2 and EP6) and a metallo-proteinase (EP7), and EP5 might be a metal-dependent serine-proteinase. Moreover, a peroxisomal polypeptide of 64 kDa was recognized by an antibody against a thiol-protease. The serine-proteinase isoenzymes (EP1, EP3 and EP4), which represent approx. 70% of the total EP activity of peroxisomes, showed a notable thermal stability, not being inhibited by incubation at 50 degrees C for 1 h.
Distefano
S
S
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apdo. 419, E-18080 Granada, Spain.
Palma
J M
JM
Gómez
M
M
Río
L A
LA
D000375:Aging; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology*; D018515:Plant Leaves; D011480:Protease Inhibitors / Q000494:pharmacology
D007527:Isoenzymes; D011480:Protease Inhibitors; D010450:Endopeptidases