Specificity of Tetrahymena calmodulin in activation of calmodulin-regulated enzymes

Abstract

Calmodulin was discovered in rat brain [ 1,2] and subsequently identified in a variety of tissues. It now appears to play a role of multifunctional intracellular Ca2+ receptor [3-51. It is thought that in general the structure and function of c~modnlin is conservatively maintained throughout the animal and plant kingdoms during evolution. We have described the isolation of calm~ulin from unicellular eukaryote, Tetrahymena pyriformis and found that it could fully activate guanylate cyclase in this organism in a Ca’+-dependent fashion [6-lo]. However, the activation of guanylate cyclase was specifically attributable to the calmodulins from Te~ra~y~ena and Paramecium [9,10]. Moreover, comparison of the primary structure of calmodulin from bovine brain [ 1 lf and Tetra~yme~a 1121 shows 12 amino acid differences between the 2 proteins. Therefore, calmodulin may vary in structure and function. Here, we have compared the functional properties between the two calmodulins from Tetrahymena and bovine brain. The data demonstrate that both Tetra~ymena and bovine brain calmodulins are similar in their potency to activate various calmodulin-dependent enzymes such as rat brain adenylate cyclase, myosin light-chain kinase, erythrocyte (Ca’+ + Mg2+)-ATPase and plant NAD kinase. Calmodulins were prepared from Tetrahymena and bovine brain as in [8]. A th~rmotolerant strain NT-l of Tetrahymena pyriformis was grown at 395°C in an enriched proteose peptone medium as in [ 131. The plasma membrane fraction which contains guanylate cyclase was prepared as in [13]. Calmodulin-dependent phosphodiesterase was prepared from bovine brain as in [14]. Adenylate cyclase was solubilized from washed particulate preparation of rat cerebrum using 1% Lubrol PX and elute from Ultro Gel AcA 34 column with 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 olo Lubrol PX, 0.1 mM EGTA, 0.1 M sucrose and 1 mM EDTA as in [15]. Myosin light-chain kinase was prepared from chicken gizzard as in [ 161. The light-chain kinase from chicken gizzard myosin was prepared essentially as in [ 171. The light chain was separated from calmodulin by DEAF&cellulose column chromatography [18], Erythrocyte membranes for (Ca2+ + Mg2+)-ATPase assay were prepared as in [ 191. Membranes were freeze-thawed 3 times just prior to incubation for ATPase assay. The preparation of NAD kinase was obtained from pea seedling (Pisum sativum) grown for 12 days in natural light at 20-25’C. The NAD kinase was prepared as in [ZO]. Guanylate cyclase activity was assayed as in [9], cyclic AMP phosphodiesterase activity as in [8]