Premium
Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography
Author(s) -
Adamíková L'ubica,
Resnick Ross J.,
Tomáška L'Ubomír
Publication year - 1996
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199607)12:9<833::aid-yea977>3.0.co;2-c
Subject(s) - affinity chromatography , biology , biochemistry , tyrosine kinase , tyrosine , receptor tyrosine kinase , protein tyrosine phosphatase , yeast , kinase , phosphorylation , saccharomyces cerevisiae , tyrosine phosphorylation , microbiology and biotechnology , enzyme , signal transduction
The direct biochemical analysis of protein tyrosine kinases from yeast has been difficult due to their very low activity in crude cell lysates. Here we present a procedure for the enrichment and partial purification of protein tyrosine kinases from Saccharomyces cerevisiae based on single‐step substrate affinity chromatography using a synthetic random co‐polymer of glutamic acid and tyrosine. Fractionation of cell lysates on a poly‐glutamic acid: tyrosine (4 : 1)‐Sepharose affinity column resulted in a 4000‐fold increase in tyrosine kinase activity. Active fractions contain at least six potential protein kinases as judged by in situ phosphorylation assay and Western blot analysis using anti‐phosphotyrosine. We propose that this protocol may also be useful for the initial identification and purification of tyrosine kinases from other organisms exhibiting low levels of this enzymatic activity in cell lysates.