Different Forms of Superoxide Dismutase from Pea Seedling Roots Differ in Sensitivity to cAMP and Calcium

Abstract

It has been established that cyclic adenosine monophosphate (cAMP), a second messenger of the adenylate cyclase signaling system, and Ca2+ are able to dose-dependently modulate the activity of various forms of superoxide dismutase (SOD) in the root cells of pea seedlings of the Rondo variety. The effect of cAMP on SOD activity in pea root cells was studied on intact seedlings by incubating their roots in 50 nM n-dibutyryl-cAMP, a fat-soluble analogue of cAMP, which led to an increase in the intracellular concentration of cAMP. Incubation of similar roots in 800 μM suramin, an inhibitor of transmembrane adenylate cyclase, contributed to a significant decrease in endogenous cAMP levels. In each of these variants, the SOD activity measured in the supernatant obtained from the root homogenate changed. Under the influence of n-dibutyryl-cAMP, the total SOD activity increased to 230%; SOD inhibitors, 3 mM KCN or 3 mM H₂O₂, added to the homogenate, reduced its activity (180 and 190% of the control, respectively). During incubation with suramin, the total activity decreased to 40% of the control value, while with the additional use of SOD inhibitors it decreased to 50–60%. Incubation of seedlings in 400 μM LaCl₃ solution resulted in a decrease in total SOD activity to 73% and in the presence of 3 mM KCN, to 56% of the control, and when 3 mM H₂O₂ was added to the homogenate, to 67%. A similar incubation of seedlings in 1 mM EGTA led to a decrease in total activity by 32%, and the inhibitors had no additional effect. The effect of calcium deficiency or excess on SOD activity was studied in a homogenate of pea seedling roots. When a calcium ion chelator, 100 mM EGTA, was added to the root homogenate, a decrease in the total SOD activity to 81% was observed; when inhibitors (H₂O₂ or KCN) were added, an even greater decrease in SOD activity occurred, up to 65 and 51%, respectively. The addition of 500 nM CaCl2 to the homogenate slightly increased the total SOD activity; KCN reduced SOD activity by approximately 20%, and H₂O₂ had no effect on this indicator. When a higher concentration of CaCl2, 500 μM, was added, the total activity did not change (100%), in the variant with KCN it decreased by 30%, and when H₂O₂ was added it remained almost unchanged. We conclude that cAMP most likely has an indirect effect on SOD activity, while calcium ions probably act directly on the active site of the enzyme molecule; Moreover, each form of SOD differs in sensitivity to calcium.