Archives of Biochemistry and Biophysics

established that Ca acts as a messenger in mediating Ca, an important intracellular messenger in plants, the action of a number of hormonal and environmental is implicated in controlling diverse cellular functions signals (3–6). Cytosolic levels of Ca increase in reby regulating the activity of several enzymes. Here we sponse to gravity, light, cold temperature, salt stress, report the presence of a Ca-dependent proteinase wind, touch, abscisic acid, auxins, and gibberellic acid (CDP) activity in roots of Arabidopsis using in-gel assays (3, 5, 6). The changes in Ca concentration are believed (zymograms). The CDP activity showed absolute Ca to regulate the activity of various enzymes through requirement for its activation; other divalent ions such Ca-modulated proteins. In plants, two Ca-moduas Mg, Sr, and Zn did not substitute for Ca in lated proteins have been well characterized (7). Calstimulating protease activity. The CDP activity was inmodulin, a multifunctional Ca-binding protein, is one hibited by the proteinase inhibitors leupeptin, E-64, and of the Ca modulated proteins. Calmodulin upon bindN-ethylmaleimide, whereas pepstatin A and phenyling Ca is able to interact with and regulate the activmethylsulfonyl fluoride were without effect. These data ity of various enzymes (3, 7). A second Ca-modulated indicate that the enzyme is likely to be a cysteine proprotein is a Ca-dependent and calmodulin-indepenteinase. The CDP activity was partially purified from dent protein kinase (CDPK) which appears to be ubiqroot cultures using ammonium sulfate precipitation, DEuitous in plants (7–9). In addition to these well charac52, Mono-Q, and Superdex 200 column chromatography. terized Ca-modulated proteins there is evidence for This purification scheme resulted in about 40-fold purithe presence of other Ca-binding proteins in plants fication of the CDP activity. Based on the elution of Arathat could be involved in sensing and mediating cytobidopsis CDP (ACDP) activity on gel filtration column solic Ca changes (10, 11). the molecular mass of CDP was estimated to be about Ca-activated neutral proteinases (also called cal75 kDa. Isoelectric focusing showed that the enzyme had pains or Ca-dependent proteinases, CDPs) are cytoa pI between 5.2 and 5.4. SDS–polyacrylamide gel analysolic proteinases that have been found in a wide variety sis showed that activity was associated with a 45-kDa polypeptide, suggesting that the native ACDP is a homoof organisms including vertebrates, invertebrates, dimer. Five different antibodies raised to animal CDPs fungi, and algae (12–22). CDPs are cysteine proteindid not cross-react with the partially purified protein. ases that have an absolute requirement for Ca for These data suggest that the plant CDP differs from the their activity (15, 16, 18). These enzymes are impliknown CDPs characterized from animals and is likely cated in regulating many cellular functions by limited to be a new CDP that is unique to plants. q 1997 Academic or complete proteolysis of other enzymes and structural