Phosphoprotein Synthesis Lab Report

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Table 2: Effect of Pronase Treatment of the Phosphoprotein Derived from the Synaptosome-Enriched Fractiona5. Treatment Total dpm in the Total dpm in the supernatant ( S ) phosphoprotein residue [S/(S + R)]  100 after digestion of after digestion the phosphoprotein ( R ) 33P 32P 33P 32P 33P 32P Pronase 180 684 30 173 86 78 Control 8 72 773 3 8 Alkaline Phosphatase 1571 886 176 109 90 89 Control 224 122 1445 1053 13 10…show more content…
The phosphorus label is definitely not adsorbed to the proteins, since the entire orthophosphate radioactivity added to the isolated synaptosomal fraction remains acid soluble. The finding that RNase does not release radioactivity from the synaptosomes-enriched fraction indicates that the preparation is not contaminated with free polyribosomes. Pronase appears to render the radioactivity from both the whole synaptosome-enriched and the final phosphoprotein residue ( Table 2 ) acid soluble, indicating that the label is initially attached to amino acids. Solubilization by alkaline phosphatase digestion proves that the phosphate is indeed covalently bound. Tryptic digestion of the final phosphoprotein residue and detection of the increased phosphorylation in the ninhydrin-staining band of peptides derived from synaptosomes of trained mice is further evidence of this. Additional proof of incorporation of radioactive phosphate into proteins was obtained by hydrolysing the phosphoprotein residue with pronase or HCl, followed by separation of the phosphoserine, phosphothreonine, and inorganic phosphate (Table 4). HCl digestion appeared to be a more effective method of hydrolysis, since more radioactivity was recovered and less extraneous ninhydrin-staining material was obtained during electrophoretic separation. The combination of Dowex column chromatography with electrophorectic separation was the best way to separate phosphoserine, phosphothreonine, and orthophosphate from

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