THE EFFECTS OF CA2+ AND CA2+.CALMODULIN ON THE DECAY OF THE INTERMEDIATES I-700(1,2) FROM NATIVE AVENA PHYTOCHROME

The decay kinetics of the microsecond I700(1,2) intermediates of 124 kDa Avena phytochrome have been studied in the presence of Ca2+, Mg2+, and Ca2+.calmodulin. Additionally, the influence of various antagonists for calmodulin-like Ca2+-binding sites on the decays has been investigated. It is shown that Ca2+ and Mg2+ are binding sufficiently to phytochrome. An increase in the concentration of each of the above cations slightly decreases the initial differential absorbance by I700i, while other cations with different ionic radii (Zn2+ and Cd2+) and charge (Al3+ and La3+) precipitate the protein already at micromolar concentrations. This confirms the high sensitivity of the ground-state far-red-light-adapted form of phytochrome (P(r)) to cations. A comparison of the primary sequence of phytochromes from different sources with sequences of EF-hand-like Ca2+-binding sites suggests a specific Ca2+-binding site. Ca2+.calmodulin strongly decreases the Arrhenius parameters of the I700(1,2) decay kinetics. This effect is inhibited by the Ca2+ antagonist 1-[bis[4-chlorophenyl]-methyl]-3-[2-(2,4-dichlorophenyl)methoxyl]ethyl-1H-imidazolium chloride (calmidazolium, compound K24571. The decrease caused by Mg+ and Ca2+ (1 mM) is smaller than that induced by Ca2+.calmodulin, and it is inhibited also by various Ca2+-binding site antagonists. The inhibitory potential of various antagonists for calmodulin-like Ca2+-binding sites is larger for Ca2+ than for Mg2+. The location for the binding site is discussed.