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Updated: Sep 3, 2026
In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity
Published on: January 5, 2016
Purification of phospholipid methyltransferase from rat liver microsomal fraction
Abstract:
Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.
Insights
Rat liver phospholipid methyltransferase exists as a 25 kDa monomer or a 50 kDa dimer. The dimer form can be phosphorylated by cyclic AMP-dependent protein kinase, suggesting distinct functional states for this key enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Phospholipid methyltransferase is crucial for synthesizing phosphatidylcholine from phosphatidylethanolamine.
- S-adenosyl-L-methionine serves as the methyl donor in this enzymatic reaction.
- Understanding the enzyme's structure and regulation is vital for cellular lipid metabolism research.
Purpose of the Study:
- To purify and characterize rat liver phospholipid methyltransferase.
- To investigate the enzyme's molecular forms and potential regulatory mechanisms.
- To determine if the enzyme can be modified by protein kinases.
Main Methods:
- Purification of phospholipid methyltransferase from rat liver microsomal fraction.
- Analysis using SDS/polyacrylamide-gel electrophoresis (SDS-PAGE) to determine molecular mass.
- Phosphorylation assays using [alpha-32P]ATP and cyclic AMP-dependent protein kinase.
- Protein identification via peptide mapping and electro-elution.
Main Results:
- Homogeneous purification revealed a single 50 kDa protein, which is phosphorylated at a specific site.
- Less purified preparations showed both 50 kDa and 25 kDa forms.
- The 25 kDa protein forms a dimer (50 kDa) and shares identical peptide maps with the 50 kDa form.
- The 50 kDa dimer is phosphorylated by cyclic AMP-dependent protein kinase.
Conclusions:
- Rat liver phospholipid methyltransferase exists in monomeric (25 kDa) and dimeric (50 kDa) forms.
- The dimeric form is susceptible to phosphorylation by cyclic AMP-dependent protein kinase.
- These findings suggest distinct functional or regulatory roles for the different molecular forms of the enzyme.
