Isolation and physico‐chemical characterization of the inhibitory protein of the troponin system

Separation and characterization of the 37 000 dalton component of the troponin system

“…CM cellulose column chromatography of crude troponin B resulted in 3 peaks [8] with the protein eluting in peak 3 corresponding to TN-T. In this investigation, it was observed that with freshly prepared eluents all the protein fractions were eluted faster and poor resolution was obtained.…”

“…Tropomyosin had no significant influence on the inhibitory effect of TN-T. For maximum inhibition it was noted that nearly 60/ag of TN-T was required per 0.2 mg of DAM, i.e. 300/zg per 1 mg, whereas with the inhibitory protein only 25/ag was required per 1 mg of DAM for a comparable extent of inhibition [8]. Since the TN-T preparations employed in this study were found to be homogeneous by SDS gel electrophoresis the observed biological activity cannot be (table 1).…”

“…Crude Troponin B prepared from rabbit skeletal muscle using the method of Hartshorne and Mueller [1 ] was further purified by CM cellulose chromatography as described earlier [8]. SDS polyacrylamide gel electrophoresis and molecular weight determinations were carried out according to Weber and Osborn [9].…”

“…Circular dichroism measurements were made on a Cary model 6001 CD attachment to a Cary 60 recording spectropolarimeter in accordance with the methodology of Mani et al [8]. Protein concentrations were determined in the ultracentrifuge employing the Rayleigh interference optical system assuming 41 fringes equivalent to 10 mg/ml [10].…”

“…CD measurements were made in 0. Low speed sedimentation equilibrium studies were carried out in a Beckman Model E analytical ultracentrifuge according to methodology described earlier [8].…”