Troponin-I is the inhibitory protein of the regulatory troponin-tropomyosin complex in striated muscle responsible for sensitising the interaction between actin and myosin to changes in calcium ion concentration. In cardiac muscle, a tissue specific isoform of troponin-I is present which possesses an additional 26 -27 residues on the N-terminus when compared with the fast and slow skeletal isoforms [I]. Two adjacent phosphoserine residues have been identified in this sequence at positions 23 and 24 [2,31. Both of these residues are substrates for CAMP and cGMP dependent protein kinases as well as protein kinase C [4]. Phosphorylation of troponin-I at these site(s) occurs in the heart in-vivo and is correlated with change in calcium sensitivity of the actin-activated myosin M& ATPase and force production [S,61. Stimulation of the heart with D-adrenergic agents also results in increased phosphorylation of troponin-I [7]. Given the cardiac specific nature of these phosphorylation sites it is important to find probes that can be used to investigate the relationship between extent of phosphorylation and biochemical function. We have therefore produced a number of monoclonal antibodies to cardiac troponin-I with this aim in mind.A panel of monoclonal antibodies directed against human and canine cardiac troponin-I were produced by conventional hybridoma techniques. Antibodies were screened by enzyme-linked immunoassay (ELISA) at an early stage for cardiac specificity and only monoclonals showing no reactivity with fast or slow skeletal isoforms were investigated. Furthermore, monoclonals were also tested for species cross-reactivities by ELISA using purified troponin-I from human, canine, porcine, bovine and rat hearts. From an original seven cardiac specific monoclonal antibodies, four (20L?G, 19/C12, I W S and B4/J36) were found to be generally species cross-reactive and were selected for further study. methods for their ability to bind to cardiac troponin-I in the myofibril. The binding of fluorescent labelled antibodies to troponin-I inside damaged cardiac myocytes was investigated by fluorescence-activated cell sorting and binding to isolated cardiac myofibrils was studied by combined phase-contrast and indirect immunofluorescence. Myocytes prepared by collagenase perfusion of isolated rat hearts contained a mixture of live and necrotic cells. These were incubated with each monoclonal antibody which was labelled with a fluoroscein isothyocyanate labelled second antibody. Non-immune mouse I& acted as a control in each cell sorting experiment and nuclear propidium iodide uptake acted as an independent fluorescent marker of cell viability. Antibodies 20nG and 19/C12 showed significant specific binding to troponin-I. B4B6 showed no binding while I W S gave intermediate binding. When tested on isolated myofibrils, the above results were confirmed with the exception that 1O/FS showed no binding. This was probably a result of the conformational state of the cross-bridges in myofibrils which were prepared under rigor condi...
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