Divalent Cation Binding to Wheat Germ Calmodulin1

Abstract

The Ca²⁺ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaC1 by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 μM⁻¹. The cysteine residue of this calmodulin is located at the 27th position from the NH₂-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9–12, Osaka). According to the quantitative analysis of the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca²⁺ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca²⁺ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mnitalic and/or Ca²⁺. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca²⁺, but was unchanged with Mn²⁺. On the other hand, Mn²⁺ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2 was estimated to be 0.8 nm. It is concluded that the first Mn²⁺ binds to a domain near the NH²⁺-terminal. The difference UV absorption spectrum induced by Mn²⁺ was similar to that induced by Ca²⁺. However, the amount of Mn²⁺ needed to saturate the difference spectrum was 1 mol more than the amount of These results indicate that the high affinity site for Mn²⁺ is in the NH₂₊-terminal half region, while the sites for Ca²⁺ are in the COOH-terminal half region.