Partial purification and characterization of a Ca(2+)-dependent proteinase from Arabidopsis roots.
Ca2+, an important intracellular messenger in plants, is implicated in controlling diverse cellular functions by regulating the activity of several enzymes. Here we report the presence of a Ca(2+)-dependent proteinase (CDP) activity in roots of Arabidopsis using in-gel assays (zymograms). The CDP activity showed absolute Ca2+ requirement for its activation; other divalent ions such as Mg2+, Sr2+, and Zn2+ did not substitute for Ca2+ in stimulating protease activity. The CDP activity was inhibited by the proteinase inhibitors leupeptin, E-64, and N-ethylmaleimide, whereas pepstatin A and phenylmethylsulfonyl fluoride were without effect. These data indicate that the enzyme is likely to be a cysteine proteinase. The CDP activity was partially purified from root cultures using ammonium sulfate precipitation, DE-52, Mono-Q, and Superdex 200 column chromatography. This purification scheme resulted in about 40-fold purification of the CDP activity. Based on the elution of Arabidopsis CDP (ACDP) activity on gel filtration column the molecular mass of CDP was estimated to be about 75 kDa. Isoelectric focusing showed that the enzyme had a pI between 5.2 and 5.4. SDS-polyacrylamide gel analysis showed that activity was associated with a 45-kDa polypeptide, suggesting that the native ACDP is a homodimer. Five different antibodies raised to animal CDPs did not cross-react with the partially purified protein. These data suggest that the plant CDP differs from the known CDPs characterized from animals and is likely to be a new CDP that is unique to plants.
Safadi
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Department of Biology, Colorado State University, Fort Collins 80523, USA.
Mykles
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Reddy
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D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008121:Nephropidae; D018517:Plant Roots / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology
D002413:Cations, Divalent; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases; D002118:Calcium