Phosphorylation-specific antibodies for human cardiac troponin-I.

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin-I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac-specific but cross-species reactive [Cummins, B., Aukland, M. L.

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998.

We extracted troponin-I (TnI) from skinned rat and rabbit soleus muscle fibres using a modification of the method described by Strauss et al. (FEBS Lett 310:229-234, 1992) for replacement of TnI in cardiac preparations. Incubation of soleus muscle fibres with 10 mmol/l vanadate virtually completely abolished the Ca2+dependence of force.

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C.

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. [Strauss, J.

The effects of phosphorylation of cardiac troponin-I on its interactions with actin and cardiac troponin-C.

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C).

Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli and their purification and characterisation. Two point mutations allow high-level expression of troponin-I.

We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria.