Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line

J. E. Casnellie, M. L. Harrison, L. J. Pike, K. E. Hellström, E. G. Krebs

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Abstract

The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [γ-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70-80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the M(r) 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60(src); the K(m) for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The K(m) for this peptide was 3-4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.

Original languageEnglish
Pages (from-to)282-286
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume79
Issue number2 I
DOIs
StatePublished - Sep 24 1982
Externally publishedYes

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