TY - JOUR
T1 - Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase
AU - Gowher, Humaira
AU - Zhang, Xing
AU - Cheng, Xiaodong
AU - Jeltsch, Albert
N1 - Funding Information:
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, JE 252/2), the Bundesminister für Bildung und Forschung (BMBF) BioFuture program, and the U.S. National Institutes of Health, Public Health Services (Grants GM49245 and GM61355 to Xing Zhang and Xiaodong Cheng). Thanks are also due to M. Schwerdtfeger for technical assistance.
PY - 2005/7/15
Y1 - 2005/7/15
N2 - Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the Km and kcat values of the Dim-5 enzyme for the methylation of a 20mer peptide to be 7.4 μM and 2.3 min-1, respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.
AB - Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the Km and kcat values of the Dim-5 enzyme for the methylation of a 20mer peptide to be 7.4 μM and 2.3 min-1, respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.
KW - Avidin-biotin
KW - Dim-5
KW - H3K9 methyltransferase
KW - Peptide methylation assay
KW - Protein methyltransferase
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U2 - 10.1016/j.ab.2005.04.028
DO - 10.1016/j.ab.2005.04.028
M3 - Article
C2 - 15935324
AN - SCOPUS:21244475895
SN - 0003-2697
VL - 342
SP - 287
EP - 291
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -