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Using Ca(2+)-dependent affinity chromatography on a synthetic compound
(W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding
proteins from rabbit lung extracts. The molecular weights of these proteins were
estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino
acid sequence of the 10-kDa protein revealed that it has two EF-hand structures.
In addition, the 10-kDa protein was highly homologous (91%) to the product of
growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the
10-kDa protein was observed by a change in the uv difference spectrum.
Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/-
0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed
to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin
light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic
protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein,
in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin
binding proteins was unique and seems to differ from that of calmodulin and
calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role
through its binding proteins for the Ca(2+)-dependent cellular response.
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