The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

Abstract

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg(2+)-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the ;molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg(2+)-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca(2+)-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg(2+)-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca(2+) in the medium. 4. The Ca(2+)-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca(2+)-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca(2+)-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.

Separation and characterization of the constituents of troponin

“…The present results clearly show that troponin preparations contain more than two protein fractions as reported by other workers [ 1,4] . Troponin A and B were found heterogenous in disc electrophoresis [5,161, and our present experiments show that troponin B contains besides peak I, the material of peaks II and III, which is probably responsible for the solubility of troponin B which differ from those of peak I.…”

“…3 and 4 show the effect of the DEAE-Sepha- dex peaks on the Mg'+-stimulated ATPase activity of DAM. The protein of peak 1 strongly inhibits ATPase activity whether or not EGTA and tropomyosin are present and in this respect it corresponds to troponin B of Hartshorne et al [l] or to the inhibitory factor of Schaub and Perry [4] (fig. 3A).…”

“…3B). Thus, the material present in this peak behaves similarly to troponin A [I] or to calcium sensitizing factor [4] . The proteins present in peak II and III also have an effect on the ATPase activity of DAM ( fig.…”

“…One of them, troponin B, inhibited the Mg*'-stimulated ATPase activity of DAM* [3] independently of the concentration of Ca2+ and of the presence of tropomyosin whereas a second fraction, troponin A, had no influence on DAM per se, but sensitized troponin B to Ca 2+ ions in the presence of tropomyosin. Two similar fractions were later obtained by Schaub and Perry [4] with the use of SE-Sephadex chromatography…”

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

“…ATPase was assayed essentially as described by Schaub and Perry [3]. The reaction mixture consisted of 1. desensitized by trypsin treatment [7].…”