Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.

• Calbindin D28k is a member of the calmodulin super-family of Ca2+ -binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca2+ buffer, but the studies presented in this work indicate that it may also act as a Ca2+ sensor. The results show that Mg2+ binds to the same sites as Ca2+ with an association constant of approximately 1.4 x 10(3) M-1 in 0.15 M KCl. The four high-affinity sites in calbindin D28k bind Ca2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg2+, the Ca2+ -affinity is reduced by a factor of two and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca2+ concentration in a resting cell calbindin D28k is saturated to 40-75% with Mg2+, but to less than 9 % with Ca2+. In contrast, the protein is expected to be nearly fully saturated with Ca2+ at the Ca2+ level of an activated cell. A substantial conformational change is observed upon Ca2+ binding, but only minor structural changes take place upon Mg2+-binding. This suggests that calbindin D28k undergoes Ca2+ -induced structural changes upon Ca2+ activation of a cell. Thus, calbindin D28k displays several properties that would be expected for a protein involved in Ca2+ -induced signal transmission and hence may function not only as a Ca2+ buffer, but also as a Ca2+ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca2+, becomes exposed upon Ca2+ binding.

Ämnesord

NATURVETENSKAP  -- Kemi -- Fysikalisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Physical Chemistry (hsv//eng)

Nyckelord

Spectrum Analysis

Animal

Amino Acid Sequence

Calcium/*metabolism

Calcium-Binding Protein

Vitamin D-Dependent/*chemistry

Cattle

Circular Dichroism

Human

Magnesium/metabolism

Models

Statistical

Molecular Sequence Data

Protein Binding

Protein Conformation

Recombinant Proteins/metabolism

Signal Transduction

Fluorescence

Spectrometry

Mass

Support

Non-U.S. Gov't

U.S. Gov't

Non-P.H.S.

Trypsin/pharmacology

Ultraviolet Rays

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