Simultaneously synthesized peptides on continuous cellulose membranes as substrates for protein kinases.
Sets of peptides with defined sequences, each on a separate spot, were synthesized simultaneously on continuous cellulose membranes (SPOTs membranes), which were originally designed for epitope studies. The applicability of the membrane-bound peptides as substrates for protein kinases was tested using protein kinase A, protein kinase C and casein kinases I and II as model enzymes. We found that the peptide-membrane complexes can serve as kinase substrates. Our results suggest that membrane-bound peptides offer a new potential for the investigation of substrate specificity of protein kinases. An advantage to this method is that there is no need for substrate identification and separation, which is required with high-volume random peptide libraries. Membrane-bound peptides may even form a basis for kinase assays with peptides lacking multiple basic amino acids, required for separation of the substrates in conventional assays. Problems connected with protein kinase substrate specificity can be investigated in any laboratory using the rapid and inexpensive SPOTs technique, as neither costly apparatus nor special experience in peptide synthesis is necessary.
Toomik
R
R
Department of Medical and Physiological Chemistry, Uppsala University, Sweden. reet.toomik@bio.embnet.se
Edlund
M
M
Ek
P
P
Obrink
B
B
Engström
L
L
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D047388:Casein Kinases; D002482:Cellulose; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D008567:Membranes, Artificial; D019151:Peptide Library; D010455:Peptides; D010748:Phosphopeptides; D012694:Serine; D002482:Cellulose; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C