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Partial purification and characterization of a protein lysine methyltransferase from plasmodia of Physarum
Abstract:
Plasmodia of Physarum polycephalum have an active protein lysine methyltransferase (S-adenosylmethionine:protein-lysine methyltransferase, EC 2.1.1.43). This enzyme has been purified 40-fold with a 13% yield, and it catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine residues with formation of N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysines in a molar ratio of 4:1:1 based on [14C]methyl incorporation into the methylated lysines. The ratio remains unchanged at all stages of the partial purification, as well as after fractionation by sucrose density gradient centrifugation and gel electrophoresis. The rate of protein methylation is time dependent, enzyme concentration dependent, and requires the presence of a sulfhydryl reducing agent for optimal activity. The enzyme has optimal activity at pH 8 and is inhibited by S-adenosyl-L-homocysteine and EDTA. Lysine-rich and arginine-rich histones serve as the most effective exogenous protein acceptors; P. polycephalum actomyosin is inactive, and chick skeletal myofibrillar proteins are 25% as effective as exogenous mixed histones as substrates. Lysine, polylysine, ribonuclease A, cytochrome c, and bovine serum albumin are not methylated.
Insights
Physarum polycephalum contains a protein lysine methyltransferase that transfers methyl groups from S-adenosyl-L-methionine to lysine residues. Histones are the most effective substrates for this enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The slime mold Physarum polycephalum exhibits enzymatic activity for protein methylation.
- Protein methylation is a crucial post-translational modification influencing protein function.
Purpose of the Study:
- To characterize the protein lysine methyltransferase in Physarum polycephalum.
- To identify the enzyme's substrates and optimal activity conditions.
Main Methods:
- Purification of the S-adenosylmethionine:protein-lysine methyltransferase.
- Enzyme activity assays using various protein substrates.
- Analysis of methylation products using radiolabeled methyl groups.
- Enzyme fractionation via sucrose density gradient centrifugation and gel electrophoresis.
Main Results:
- The enzyme was purified 40-fold, catalyzing the formation of mono-, di-, and trimethyllysines in a 4:1:1 ratio.
- Optimal enzyme activity was observed at pH 8, requiring a sulfhydryl reducing agent.
- Lysine-rich and arginine-rich histones were identified as the most effective protein acceptors.
- Actomyosin and other proteins were not methylated, indicating substrate specificity.
Conclusions:
- Physarum polycephalum possesses a specific protein lysine methyltransferase.
- Histones are preferred substrates, suggesting a role in chromatin regulation.
- The enzyme's activity is dependent on specific conditions and reducing agents.