Abstract
Bovine cardiac troponin-I (TN-I) and troponin-T (TN-T) have been examined in solution using ultracentrifugation, gel filtration, and viscosity. A new method of purifying TN-T, employing hydroxylapatite chromatography in 6 M urea, is reported. Cardiac TN-T (Mr = 36,000) undergoes a reversible, concentration-dependent association in nondenaturing buffers, probably to a tetramer. The Stokes radius (Rs) of aggregated TN-T, determined by sedimentation velocity and gel chromatography on Sephacryl S-300, is 80 A and the reduced viscosity of the subunit ranges from 20 to 25 ml/g at protein concentrations between 0.5 and 2.5 mg/ml. These data suggest that TN-T forms highly asymmetric aggregates in solution. Bovine cardiac TN-I also has a tendency toward self-association, but is essentially monomeric (Mr = 23,000) at protein concentrations below 1 mg/ml. The presence of reducing agent is necessary to avoid intermolecular disulfide bond formation. From gel filtration experiments, the value of Rs is 29 A indicating that TN-I is a moderately asymmetric protein (frictional ratio = 1.5). Similar properties are observed when both sulfhydryl groups of TN-I are modified by carboxamidomethylation.
Highlights
36,000) undergoes a reversible, concentration-depend- a tendency to aggregate (Burtnick et al, 1975, 1976)
= 23,550) than rabbit skeletal TN-I, containing an extra 26 amino acidresidues at the NH2 terminus.This difference appears to befunctionally significant since the phosphorylation of a serine in this region,which is under hormonal control, decreases the sensitivityof troponin regulation toCa2+(Solar0et aZ.,1976).The molecular weights and amino acid compositions of rabbit and bovine cardiac TN-I
The results suggest that both TN-I and TN-T are asymmetmrioclecules
Summary
36,000) undergoes a reversible, concentration-depend- a tendency to aggregate (Burtnick et al, 1975, 1976). Bovine cardiac TN-I has a tendencytoward self-association, but is essentially monomeric (Mr = 23,000) at protein concentrations below l mg/ml. Troponinfromskeletal muscle has Protein Preparation-Bovine cardiac troponin subunits were lated from crude troponin (Tsukui and Ebashi, 1973) by DEAE-
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