Abstract
Phosphatidylethanolamine N-methyltransferase catalyzes the synthesis of phosphatidylcholine from phosphatidylethanolamine and is most active in liver. A cDNA for this enzyme from a rat liver cDNA library has been cloned, sequenced, and expressed in COS-1 cells, McArdle-RH7777 rat hepatoma cells, and Sf9 insect cells. The expressed protein was capable of converting phosphatidylethanolamine into phosphatidylcholine in intact COS-1 cells, which normally have very low methyltransferase activity. The calculated molecular mass of the methyltransferase protein is 22.3 kDa, which is equivalent to that of the pure protein isolated from rat liver. Comparison of the sequence of the cloned rat liver methyltransferase with the yeast phosphatidylethanolamine methyltransferase PEM2 gene product revealed 44% identical amino acids and 68% similarity in the two predicted protein sequences. A polyclonal antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal region of the enzyme and was affinity purified. The antibody recognized a single protein with a molecular mass of approximately 20 kDa when either rat liver proteins or proteins derived from the transfected COS-1 cells were electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate. Surprisingly, the antibody exhibited no reactivity with endoplasmic reticulum proteins, even though the major phosphatidylethanolamine methyltransferase activity resides on this subcellular organelle. Instead, the antibody specifically recognized a protein in a unique subcellular membrane fraction purified from a crude mitochondrial preparation on a Percoll gradient. Immunocytochemical examination by electron microscopy showed positive labeling only in unique regions of the hepatocytes. The data suggest that this phosphatidylethanolamine methyltransferase is a specific marker for this unique membrane fraction.
Highlights
Catalyzes the synthesis of phosphatidylcholine from is an enzyme in animals that converts PE to PC and uses phosphatidylethanolamine and ismost active in liver
The has been cloned, sequenced, and expressed in COS-1 majority of PEMT activity is localized to thecytosolic surface cells, McArdle-RH7777 rat hepatoma cells, and Sf9 of the ER(Ridgway,1989).The enzyme was purifiedfrom rat insect cells
The expressed protein was capabolfecon- liver in 1987 (Ridgwayand Vance, 1987)and has been shown verting phosphatidylethanolamine into phosphatidyl- to exist as a single protein with a molecular mass of 19 kDa
Summary
CDNA Cloning and Sequencing-The NH2-terminal sequence (30 amino acid residues) of purified rat liver PEMT was obtained by Edman degradation (Ridgway, 1988). The insert was 500 base pairs in length and was potentially a partial fragmenotf rat liver PEMT cDNA according to analysis of its open reading frame. The livers were perfused with ice-cold phosphate-buffered saline through the portal vein and rapidly removed,and subcellular fractions (ER1 and ER2 fractions, Golgi membranes, plasma membrane, mitochondria and nuclei) were isolated according to modifications (Vance, 1990)of the procedure of Croze and Morri. The open reading frame encodes a 199-amino acid protein mitochondria was removed from approximatelytwo-thirds down the tube Another membrane fraction, previously designated as fraction X (Vance,1990),was isolated from the Percoll gradientas the white band immediately above the mitochondria.
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