Characterization of the cardiac troponin I phosphorylation domain by 31P nuclear magnetic resonance spectroscopy.
Cardiac holotroponin can be phosphorylated at serine 23 and/or 24 in the heart-specific region of bovine troponin I. When isolated freshly it is composed of a mixture of non-, two mono-, and bisphosphorylated species. At neutral pH the monophosphorylated form carrying phosphate at serine 24 yields a resonance signal at 4.6 ppm and that carrying phosphate at serine 23 at 4.4 ppm; the two phosphate groups of the bisphosphorylated form yield only one 31P-NMR signal at 4.2 ppm. From the chemical shift dependence on pH, pKa values have been estimated to be 5.3 and 5.6 for the phosphate groups at serine 24 and serine 23, respectively. Both phosphates of the bisphosphorylated form exhibit very similar pKa values of approximately 5.8. Separation of bisphosphotroponin I from the complex results in a downfield shift and the appearance of two 31P-NMR signals at positions comparable to those of the two monophospho forms. Complex formation of cardiac troponin I with C or T does not alter the spectrum obtained with isolated troponin I; however, the original troponin spectrum is restored by reconstitution of the holocomplex from all three components T, I, and C. Two signals are also observed with a bisphosphorylated synthetic peptide [PVRRRS(P)S(P)ANYR] representing the phosphorylation domain. pKa values of about 5.3 and 5.6 have been determined for serine 7 (corresponding to serine 24 of troponin I) and serine 6 of the peptide (corresponding to serine 23 of troponin I).
Jaquet
K
K
Krankenhausbetriebsgesellschaft mbH, Herzzentrum Nordrhein-Westfalen, Bad Oeynhausen, Germany.
Korte
K
K
Schnackerz
K
K
Vyska
K
K
Heilmeyer
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D010759:Phosphorus Isotopes; D010766:Phosphorylation; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I
D009842:Oligopeptides; D010759:Phosphorus Isotopes; D014336:Troponin; D019210:Troponin I