title: Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography.
abstract: 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.
authors.affiliation: Department of Biochemistry, Comenius University, Faculty of Natural Sciences, Bratislava, Slovakia.
authors.forename: L
authors.initials: L
authors.lastname: Adamíková
authors.forename: R J
authors.initials: RJ
authors.lastname: Resnick
authors.forename: L
authors.initials: L
authors.lastname: Tomáska
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods*; D005973:Glutamine; D010766:Phosphorylation; D011108:Polymers; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012685:Sepharose; D014443:Tyrosine
chemical_list: D000596:Amino Acids; D011108:Polymers; C080178:glutamine tyrosine copolymer; D005973:Glutamine; D014443:Tyrosine; D012685:Sepharose; D011505:Protein-Tyrosine Kinases