Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane.
Tetrahymena calmodulin (CaM) differs from mammalian CaM in its ability to activate Tetrahymena guanylate cyclase. Of 12 differences in amino acid sequence, two occur near the carboxyl terminus (Gln-143----Arg and Thr-146----deletion). To investigate the functional significance of the carboxyl-terminal region in activation of the guanylate cyclase, three mutated CaMs were engineered by using cassette mutagenesis of rat CaM cDNA: Gln-143----Arg (CaM.A), Thr-146----deletion (CaM.D), and Gln-143----Arg/Thr-146 deletion (CaM.AD). Recombinant wild type CaM (wCaM), CaM.A, CaM.D, and CaM.AD were indistinguishable in their ability to activate cyclic AMP phosphodiesterase. The two mutated CaMs (CaM.A and CaM.AD) with the Gln-143 replacement activated guanylate cyclase of Tetrahymena plasma membrane in the presence of Ca2+, with the maximal activation being half of that produced by Tetrahymena CaM. In contrast, neither CaM.D nor wCaM could stimulate the cyclase activity. A CaM antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), prevented the cyclase activation by either Tetrahymena CaM, CaM.A, or CaM.AD. Thus, we conclude that Arg-143 is in a region of the molecule involved in activation of Tetrahymena guanylate cyclase. The data also suggest that the cyclase activation by Tetrahymena CaM requires complex macromolecular interactions between the entire CaM molecule and the enzyme.
Nagao
S
S
Department of Biochemistry, Gifu University School of Medicine, Japan.
Matsuki
S
S
Kanoh
H
H
Ozawa
T
T
Yamada
K
K
Nozawa
Y
Y
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000235:genetics* / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004274:DNA, Recombinant / Q000378:metabolism; D004789:Enzyme Activation; D005821:Genetic Techniques; D005973:Glutamine; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D013768:Tetrahymena / Q000201:enzymology*
D002147:Calmodulin; D004274:DNA, Recombinant; D005973:Glutamine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006162:Guanylate Cyclase