Abstract
Abstract Purified troponin prepared according to published procedures was separated into four major protein fractions when chromatographed on DEAE-Sephadex in 6 m urea. The main components in Fractions 1 to 4 had approximate molecular weights of 14,000, 24,000, 35,000, and 21,000, respectively, as determined by sodium dodecyl sulfate gel electrophoresis. Reconstitution experiments using all possible combinations indicated that Fractions 2, 3, and 4 restored the Ca++ requirement for the ATPase activity of actomyosin in the presence of tropomyosin. Recovery of activity could only be achieved when the required components were mixed prior to removal of urea. This suggests that one or more of these proteins may refold improperly on removal of urea unless the other components are present.
Highlights
Assay of Troponin Activity-Troponin activity was measured by determining the EGTA inhibition of actomyosin ATPase activity in the presence of tropomyosin
An additional advantage of electrophoresis in SDS gels is the good correlation between the mobility of the proteins and the molecular weights [22, 23]
It should be noted that Fractions 1, 2, and 3 from the DEAE-Sephadex column all showed essentially the same diffuse electrophoretic band patterns in the 8 M urea gels (Fig. 3)
Summary
Protein Determination-Protein concentrations were deter mined by the biuret reaction [18], which had been standardized with troponin on the basis of micro-Kjeldahl analyses, assuming a16% nitrogen content.Assay of Troponin Activity-Troponin activity was measured by determining the EGTA inhibition of actomyosin ATPase activity in the presence of tropomyosin.Assays were conducted at 25” and pH 7.5 in a medium containing 25 mM Tris, 25 mu KCl, 5 mM MgCL, 5 mu ATP, and 0.01 mM CaClz or 1 mu EGTA in a volume of 2 ml. Protein Determination-Protein concentrations were deter mined by the biuret reaction [18], which had been standardized with troponin on the basis of micro-Kjeldahl analyses, assuming a. Assay of Troponin Activity-Troponin activity was measured by determining the EGTA inhibition of actomyosin ATPase activity in the presence of tropomyosin. Assays were conducted at 25” and pH 7.5 in a medium containing 25 mM Tris, 25 mu KCl, 5 mM MgCL, 5 mu ATP, and 0.01 mM CaClz or 1 mu EGTA in a volume of 2 ml. The reaction was initiated with ATP and terminated after 5 min by addition of 1 ml of 20% trichloroacetic acid. The usual specific activity of the actomyosin ATPase was of the order of 0.2 pmole per min per mg in either the calcium or EGTA medium
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