CLONING, RECOMBINANT EXPRESSION AND CHARACTERIZATION OF WILD TYPE-105-TRP-CALMODULIN OF THE GREEN-ALGA MOUGEOTIA-SCALARIS

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the cDNA inserted into the prokary otic expression vector pGEX-2T. The recombinant calmodulin protein (Ca M) was expressed as a fusion product together with glutathione S-trans ferase and isolated on glutathione sepharose. After cleavage and purif ication, the CaM was characterized by Ca2+-dependent shift in SDS-PAGE , by activation of cyclic 3:5' nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca2+ buffers in the PDE test, a ffinity to Ca2+ of Mougeotia CaM was found to be diminished fivefold c ompared to maize or bovine brain CaMs. There was also a 20-fold increa se of half maximal activation (K-act) in the PDE test for Mougeotia Ca M relative to maize CaM, while the K-act of maize CaM to that of bovin e brain CaM was almost the same. The derived amino acid sequences of C aM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) --> Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.