FIG. 3.

The <10-kDa nonprotein component was purified and identified as 5′-AMP. (A) The <10-kDa cardiocyte fraction was chromatographed on a Pharmacia PepRPC column (graph); fractions (Fr.) showing inhibitory activity (fractions 9 and 10) on c-Src autophosphorylation in the presence of the >10-kDa fraction are shown in the autoradiograph. (B) PepRPC inhibitory fractions were pooled and chromatographed on a Pharmacia Superdex column (graph). A major peak fraction (fraction 42) exhibiting combinatorial inhibitory effect on c-Src autophosphorylation (autoradiograph) corresponds to a molecular mass of 350 Da. The column was precalibrated as described in Materials and Methods. (C) Molecular mass determination of the purified fraction from a Superdex column (top) and 5′-AMP with purity of >99% (bottom) was performed by ESI-mass spectroscopy. The molecular mass of the purified sample was determined to be 347.8 Da, matching that of 5′-AMP. (D) 1H NMR spectra of the purified fraction from the Superdex column (top) and 5′-AMP (bottom) were determined as described in Materials and Methods. In the NMR profiles, dimethyl sulfoxide (DMSO) and H₂O lines are marked, and an unmatched minor line in the purified sample is indicated by an asterisk. (E) Combinatorial c-Src inhibitory activity due to the addition of increasing amounts of either the purified Superdex column fraction (sample) or 5′-AMP, measured in the presence of cardiocyte >10-kDa fraction (0.1 μg/μl, final concentration). Nucleotide concentrations (Conc) in the purified <10-kDa fraction and in 5′-AMP samples were adjusted on the basis of absorbance at 259 nm. c-Src autophosphorylation was quantitated by Cerenkov counting (graph). (F) Inhibitory effect by the combined action of 5′-AMP and the >10-kDa fraction in assays using other nucleotide analogs. c-Src autophosphorylation was measured with increasing concentrations of AMP isomers and related nucleotides. In the absence (−) of the >10-kDa fraction, the effect on c-Src activity was measured with two higher concentrations (0.08 and 0.16 μg/μl) of nucleotides. ³²P-labeled c-Src was detected by autoradiography.