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N. D. Ridgway, D. E. Vance Purification of Phosphatidylethanolamine N-Methyltransferase from Rat Liver.
J. Biol. Chem.1987. Vol. 262; , 17231– 17239.
Z. Yao, D. E. Vance The Active Synthesis of Phosphatidylcholine Is Required for Very Low Density Lipoprotein Secretion from Rat Hepatocytes.
J. Biol. Chem.1988. Vol. 263; , 2998– 3004.
Z. Cui, J. E. Vance, M. H. Chen, D. R. Voelker, D. E. Vance Cloning and Expression of a Novel Phosphatidylethanolamine N-Methyltransferase. A Specific Biochemical and Cytological Marker for a Unique Membrane Fraction in Rat Liver.
J. Biol. Chem.1993. Vol. 268; , 16655– 16663.
C. J. Walkey, L. Yu, L. B. Agellon, D. E. Vance Biochemical and Evolutionary Significance of Phospholipid Methylation.
J. Biol. Chem.1998. Vol. 273; , 27043– 27046.
In the 1980s and '90s, Dennis Vance and colleagues at the University of Alberta published a series of papers demonstrating the importance of the phospholipid phosphatidylcholine (PC) to the assembly and secretion of lipoproteins from the liver. According to William Dowhan at the University of Texas Health Science Center at Houston, these studies represented “an entry into the evolving area of molecular biology” in the field of mammalian lipid enzymology.
PC is a major membrane phospholipid found in eukaryotic cells. It is a major component of the membrane bilayers of all cellular organelles. PC is also an integral part of the monolayers surrounding certain plasma lipoproteins, such as high density lipoproteins (HDL), or “good” cholesterol; low density lipoproteins (LDL), or “bad” cholesterol; and very low density lipoproteins (VLDL). VLDL is assembled in the liver and secreted into the bloodstream, where it is a precursor to LDL; without PC, the lipids can remain behind and build up in the cell.
PC is formed by two pathways: the choline pathway, which is found in all mammalian tissues and uses the dietary nutrient choline as its precursor; and the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, which starts with methionine and operates almost exclusively in the liver.
By 1987, Vance had been working for many years to understand the regulation of these two pathways. His lab had been trying to purify the enzyme PEMT since 1976. Membrane-bound enzymes are notoriously difficult to purify because they do not dissolve in aqueous buffers. A detergent was needed to solubilize the enzyme, which meant that normal purification methods, such as column chromatography, had to be modified. Once the enzyme was solubilized in buffer, it would very quickly lose activity. For these reasons, Vance refers to the purification of membrane proteins as “masochistic enzymology.”
Neale Ridgway, who was a doctoral student in Vance's lab, was able to accomplish this feat in 1987. Vance says Ridgway “had this desire to purify the enzyme, and he didn't give up.” Ridgway had a stroke of inspiration one day when he decided to use a phosphate rather than a Tris buffer in the purification process. He succeeded in stabilizing and then purifying the enzyme. “Thus PEMT became one of the first membrane-bound enzymes to be purified from mammalian sources,” says George Carman at Rutgers University, a JBC associate editor. “This was a miraculous feat that Ridgway had performed,” he says. “It was a major accomplishment in the field of lipid metabolism.”
Next, Vance and colleagues cultured rat liver cells to understand the relative importance of the two pathways to PC production. They fed rats a choline-free diet to hinder production of PC by the choline pathway. They then cultured cells from the rats' livers and added back choline or methionine. They found that both pathways were needed to secrete normal amounts of VLDL into the circulation. The studies “provided an important link between the liver phospholipid metabolism and lipoprotein production,” says Dowhan.
Vance's lab then worked for several years to clone the cDNA that encodes PEMT. “These days, you can isolate a gene almost at will using bioinformatics,” Carman says. “But back then, it was not trivial.”
Using sequences from the cDNA, by 1998 they had developed a knockout mouse that was unable to make PC via the PEMT pathway. These mice appeared normal until choline was removed from their diet, hindering PC production via the choline pathway.
Without choline, the rats' cells were unable to secrete VLDL. The rats developed liver failure after only a few days. The comparison of a healthy rat liver with that of a PEMT knockout mouse was striking. Vance and his colleagues had shown how important the PEMT pathway is when the choline pathway is impaired. “It was totally unexpected and totally amazing at the time,” Vance recalls. “Nobody could believe it.”
Vance's recent work has focused on physiological roles for PEMT. He is examining the implications of the enzyme for obesity and type 2 diabetes. Only by powering through the tedious tasks of extracting PEMT and isolating the gene were Vance and his team able to lay the foundation for this later work. The 1998 paper, Carman says, “could never have been done if they didn't purify the enzyme in the first place in 1987.”