Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography.
Adamikova L, Resnick RJ, Tomaska L
Yeast (1996)
Category: methods, yeast-misc ¤ Added: Aug 21, 2004 ¤ Rating: ◊◊
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.