MICROANALYSIS AND DISTRIBUTION OF CARDIAC TROPONIN-I PHOSPHO SPECIES IN HEART AREAS

Sequential phosphorylation and dephosphorylation of cTnl by the cAMP d ependent protein kinase and by protein phosphatase 2A, respectively, p roduce the non-, mono- and bisphosphorylated species (Jaquet et al., 1 995, Eur. J. Biochem. 231, 486-490). The aim of this study was to dete rmine these forms even in small tissue samples, e.g. in biopsy probes of similar to 30 mg which would allow to define the phosphorylation st ate of cTnl in heart areas. In order to do so a micro isolation proced ure for cTnl had to be established. cTnl is extracted from small bovin e, rabbit and human heart tissue samples (30-100 mg) under special con ditions avoiding dephosphorylation and is isolated by affinity chromat ography on cTnC Sepharose, All three species, the bis-, mono- and deph ospho cTnl, are precipitated quantitatively by acetone, then they are separated by non-equilibrium isoelectric focussing and quantified by s canning densitometry. The method presented here allows to quantify the three cTnl species reproducibly. No other phosphorylated species are detected, Truncated cTnl forms of each phospho species are found in hu man biopsy samples due to removal of a similar to 36 amino acid peptid e from the C-terminus. In bovine, human and rabbit heart the pattern o f the three cTnl phospho species is characteristic for left and right atrium, left and right ventricle and septum.