Jennifer E. Van Eyk

A synthetic peptide mimics troponin I function in the calcium‐dependent regulation of muscle contraction

A new technique for treating skinned cardiac muscle fibers has been developed in which troponin I is extracted, giving rise to unregulated fibers. The effect of the 12‐residue troponin I peptide on these fibers indicates that this region of troponin I is solely responsible for muscle relaxation (inhibition of force). Furthermore, troponin I peptide‐troponin C reconstituted fibers are stable through several contraction‐relaxation cycles indicating the peptide can switch binding sites between actin and troponin C. The troponin I peptide can substitute for the native protein as part of the calcium‐sensitive molecular switch that controls muscle regulation.

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

Separation of basic peptides by cation-exchange high-performance liquid chromatography

Chromatographic separations of a series of highly basic peptides on commercially available 300—A pore size CM 300 weak cation-exchange columns have been compared at various loads, pHs and ionic strengths of the eluent. On analytical columns (250 x 4.1 mm I.D.), mixtures of basic peptides containing 7 – 9 nmole of each component were separated with a 50 mM KH2PO4–KC1 gradient (pH 4.5) and under isocratic conditions (pH 4.5 and 6.5). The isocratic conditions demonstrated the effects of pH and ionic strength on retention time and resolving power on the CM 300 column. The load capacity of a CM 300 preparative column (250 x 10 mm I.D.), studied under gradient conditions (50 mM KH2PO4, 0.2 – 0.4 M KC1, pH 4.5 and 6.5), revealed that its capacity is much greater at pH 6.5. Loads up to 10–20 mg (6.6–13.3 μmol) could be applied before peaks in the crude peptide sample tested were seen to fuse.

Studies on the regulatory complex of rabbit skeletal muscle: Contributions of troponin subunits and tropomyosin in the presence and absence of Mg2+ to the acto-S1 ATPase activity

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg2+ on the acto-S1 ATPase have been examined. The effect of free Mg2+ on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg2+ concentrations: 2.5 mM Mg2+-2.5 mM ATP, conditions considered to have low free Mg2+; and 5.0 mM Mg2+-2.5 mM ATP, conditions providing a high free Mg2+ concentration of ∼2.5 mM. In the presence of high free Mg2+ (2.5 mM ATP-5.0 mM MgCl2) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40–50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg2+ on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg2+ there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg2+. Similarly, in the presence of TM and Tn, Mg2+ exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg2+. Furthermore, ultracentrifugation binding studies using14C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg2+ concentrations, indicating that faciliation of binding to F-actin by Mg2+ is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg2+ binding and by analogy Ca2+ binding to the Ca2+-Mg2+ sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca2+ binding to the Ca2+-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca2+. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca2+, indicate that the role of TnT involves more than anchoring Tn to the thin filament.

Myosin and Troponin Peptides Affect Calcium Sensitivity of Skinned Muscle Fibres

It is well known that the force developed by the contractile system depends on the calcium ion concentration in the medium surrounding the myofilaments as well as on the Ca++-sensitivity or the calcium responsiveness of the contractile apparatus (Ruegg 1988). We have studied the effect of peptides derived from troponin-I and the 20kDa domain of myosin subfragment-1 which affect the calcium sensitivity of the contractile system. These effects are of particular interest in the case of cardiac muscle, where the relationship between intracellular calcium concentration and force may vary over a wide range. For instance, hearts that are subjected to long periods of ischemia may develop a contracture even at basal levels of free calcium (Allshire et al. 1987, Allen and Orchard 1987). This is probably due to a potentiation of ATPase and force generation occurring at low levels of ATP (Winegrad 1979). Indeed, Guth and Potter (1987) showed, that attached crossbridges increase the calcium responsiveness of skinned fibres at low ATP-concentrations, when some crossbridges are in the nucleotide-free state. Under these conditions, the contractile system is activated, and even potentiated at very low calcium ion concentrations which normally do not elicit a contraction (cf. Weber and Murray 1973). In vitro, such a potentiation also occurs in the presence of excess of myosin subfragment-1, S1, indicating that there is cooperativity in the binding of S-1 to actin (Greene and Eisenberg 1980). Here we propose as a working hypothesis that it may be the actin-binding region of subfragment-1 around the SH-1 group which by, interacting with actin, is capable of “turning on” actin, so that calcium responsiveness increases.