JBC, Vol. 251, Issue 20, 6315-6319, Oct, 1976
Bovine adrenal medulla troponin-C. Demonstration of a calcium-dependent conformational change
I. C. Kuo and C. J. Coffee
A high affinity calcium-binding protein has recently been purified from the
adrenal medulla (Kuo, I.C.Y., and Coffee, C.J. (1976) J. Biol. Chem. 251,
1603-1609). This protein is closely related in its chemical and physical
properties to troponin-C (TN-C) of muscle tissue. Further examination of
the adrenal medulla protein indicates that the removal of calcium is
accompanied by a marked change in the conformation. This change in
structure is similar, if not identical, to the calcium-dependent
conformational change which has been described for skeletal muscle TN-C
(Murray, A.C., and Kay, C.M. (1972) Biochemistry 11, 2622). The far
ultraviolet circular dichroism spectrum of native adrenal medulla
calcium-binding protein (AM-CBP) shows characteristic helical ellipticity
bands at 222 and 207 nm. The helical content, as estimated from these data,
is between 40 and 45%. Removal of calcium is accompanied by a change in
ellipticity which corresponds to a decrease from 40 to 20% in the helical
content. The near-ultraviolet circular dichroism spectrum shows negative
dichroic bands at 262 and 268 nm which are characteristic of phenylalanine.
These bands are relatively insensitive to changes in the calcium ion
concentration. Sedimentation velocity studies likewise are indicative of a
calcium-dependent structural alteration. The sedimentation coefficient of
the native protein was observed to be 1.89 S. Similar measurements
performed in the presence of 3 mM ethylene glycol bis(beta-aminoethyl
ether) N,N, N', N'-tetraacetic acid (EGTA) gave a sedimentation coefficient
of 1.50 S. The molecular weight, as determined by sedimentation equilibrium
studies, was 16,000 regardless of whether the measurements were made in the
presence of CaCl2 or EGTA. From the elution properties of AM-CBP on
Sephadex G-100, the Stokes radius was observed to be 19.8 A in the presence
of calcium and 21.9 A in the presence of EGTA. All of these changes which
were induced by the addition of EGTA were completely reversible by the
readdition of excess CaCl2. These data suggest that the removal of calcium
from AM-CBP is accompanied by a pronounced conformational change which
occurs without a molecular weight change. The decreased sedimentation
coefficient, the increased Stokes radius, and the reduced helical content,
which are observed for the apoprotein, indicate that removal of calcium
results in a transformation from a compact symmetrical structure to one
that is less ordered and more asymmetrical.
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