J. Biol. Chem., Vol. 265, Issue 11, 5926-5929, Apr, 1990
Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane
S Nagao, S Matsuki, H Kanoh, T Ozawa, K Yamada and Y Nozawa
Department of Biochemistry, Gifu University School of Medicine, Japan.
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.
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