Biochemical Comparison of Tpm1.1 (α) and Tpm2.2 (β) Tropomyosins from Rabbit Skeletal Muscle.

Elke M Lohmeier-Vogel, David H Heeley

Biochemistry 2016 Mar 8

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Tpm1.1 (α) and Tpm2.2 (β) tropomyosins (39 amino acid substitutions) were isolated from adult rabbit skeletal muscle without chemical modification of cysteine, with negligible phosphorylation as assessed by two-dimensional polyacrylamide gel electrophoresis, and characterized biochemically. Reconstituted skeletal thin filaments composed of Tpm2.2 produce ∼30% less Ca(II)-induced activation of the steady-state actomyosin-S1MgATPase rate than Tpm1.1 does. This is observed at a high S1/actin ratio (6 μM myosin-S1A1, 3 μM thin filaments, pCa 4) and as a function of pCa (0.3 μM myosin-S1A1, 25 μM thin filaments). The two pCa versus MgATPase relationships are similar in terms of their steepness and midpoint. Isotype has a bearing on self-polymerization and interaction with troponin. Solutions (pH 7, ionic strength of ∼30 mM) of Tpm2.2 are more viscous than solutions of Tpm1.1, an observation explained by substitutions at the carboxy-terminal end of the molecule, including His276Asn and Met281Ile. Conversely, the enhancement of viscosity of Tpm1.1 by skeletal troponin is greater than that for Tpm2.2. Further, Tpm1.1 binds more strongly than Tpm2.2 to skeletal troponin-Sepharose, as evidenced by a later elution position in the salt gradient. Mixtures of tropomyosin and the amino-terminal CNBr fragment of troponin-T, CB1 (residues 1-151), were chromatographed on a size exclusion column in the presence of different concentrations of KCl. In 0.1 M salt, CB1 co-elutes with either isoform but is largely dissociated at 0.22 M. At intermediate salt concentrations, different degrees of complexation are observed, more extensive for Tpm1.1 than for Tpm2.2. Thus, the first reported variants of tropomyosin are distinct in their interactive and functional properties. The biochemical properties of Tpm2.2 are of particular relevance to the immature skeletal muscle thin filament.

Citation

Elke M Lohmeier-Vogel, David H Heeley. Biochemical Comparison of Tpm1.1 (α) and Tpm2.2 (β) Tropomyosins from Rabbit Skeletal Muscle. Biochemistry. 2016 Mar 8;55(9):1418-27