Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography

✍ Scribed by Adamíková, L'ubica; Resnick, Ross J.; Tomáška, L'Ubomír

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 471 KB
• Volume: 12
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(199607)12:9<833::aid-yea977>3.0.co;2-c

✦ Synopsis

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-glutaniic acid : tyrosine (4 : I)-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.