A potential pitfall in protein kinase assay: phosphocellulose paper as an unreliable adsorbent of produced phosphopeptides.
The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.
Toomik
R
R
Laboratory of Bioorganic Chemistry, Tartu University, Estonia.
Ekman
P
P
Engström
L
L
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D013552:Swine
D010748:Phosphopeptides; D010750:Phosphoproteins; D002482:Cellulose; C008881:phosphocellulose; D011494:Protein Kinases; D011493:Protein Kinase C