Biomedical subjects

J E Vance

Publications and source records attributed to J E Vance.

At least 73 records · Page 4Linked to original sources

PubMed1988

Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion?

Phosphatidylcholine and phosphatidylethanolamine in lipoproteins secreted from cultured rat hepatocytes are derived from specific biosynthetic pools (Vance, J. E., and Vance, D. E. (1986) J. Biol. Chem. 261, 4486-4491). We have tested the hypothesis that some of the phospholipids destined for secretion with lipoproteins may be made in the Golgi. Golgi fractions were prepared by three different procedures. Although each procedure yielded membranes highly enriched in galactosyltransferase, the protein profiles on polyacrylamide gels were distinct for each preparation. Similarly, the presence of phospholipid synthetic enzyme activities differed among the preparations of Golgi. Two of the preparations were judged to be contaminated by no more than 15% with endoplasmic reticulum. Although an unequivocal conclusion that Golgi contains phospholipid biosynthetic enzymes is not possible, the available evidence is consistent with this hypothesis. Golgi prepared by one method (Croze, E. M., and Morré, D. J. (1984) J. Cell. Physiol. 119, 46-57) was studied in detail. This preparation contained activities for CTP:phosphocholine cytidylyltransferase, CDP-choline:1,2-diacylglycerol cholinephosphotransferase, CDP-ethanolamine:1,2-diacylglycerol ethanolamine-phosphotransferase, phosphatidylethanolamine-N-methyltransferase, and phosphatidylserine synthase. These enzyme activities in the Golgi displayed properties similar to the enzyme activities in endoplasmic reticulum with respect to Km values for substrates, pH optima, cofactor requirements, and inhibition by metabolites. Topology experiments suggested that these enzymes on endoplasmic reticulum and Golgi are all exposed to the cytosolic surface. Phosphatidylserine decarboxylase was not detected in the Golgi preparation. The results support the hypothesis that Golgi has the capacity to make certain phospholipids for lipoprotein secretion: phosphatidylcholine via the CDP-choline and methylation pathways, phosphatidylethanolamine by the CDP-ethanolamine pathway, and phosphatidylserine. Synthesis of phosphatidylethanolamine via decarboxylation of phosphatidylserine does not appear to occur in Golgi.

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PubMed1986

A deazaadenosine-insensitive methylation of phosphatidylethanolamine is involved in lipoprotein secretion.

We have examined the effect of inhibitors of methylation of phosphatidylethanolamine on lipoprotein secretion from cultured rat hepatocytes. The incorporation of [1-3H]ethanolamine into phosphatidylcholine of hepatocytes and secreted lipoproteins was inhibited by greater than 90% by the methylation inhibitors 3-deazaadenosine and Neplanocin. In addition, these compounds strongly inhibited the incorporation of [3-3H]serine into the choline moiety of phosphatidylcholine of the hepatocytes, but had no effect on incorporation of [3-3H]serine into secreted phosphatidylcholine. The results suggest that a pool of phosphatidylcholine targeted for lipoprotein secretion originates from phosphatidylethanolamine made from serine and this methylation reaction has the unique property of being insensitive to 3-deazaadenosine.

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PubMed1986

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes.

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.

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PubMed1986

The biosynthesis of phosphatidylcholine by methylation of phosphatidylethanolamine derived from ethanolamine is not required for lipoprotein secretion by cultured rat hepatocytes.

The role that phosphatidylcholine biosynthesis plays in the assembly and secretion of lipoproteins has been investigated in rat hepatocytes, since phosphatidylcholine is the major phospholipid in all serum lipoproteins. Phosphatidylcholine in rat hepatocytes can be made via the CDPcholine pathway or by the methylation of phosphatidylethanolamine. A specific inhibitor of cellular transmethylation, 3-deazaadenosine (10 microM), has been incubated with rat hepatocytes, and we have shown that the biosynthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine derived from ethanolamine was inhibited by greater than 95%. However, incubation of 3-deazaadenosine with cultured rat hepatocytes for up to 18 h did not affect the secretion of any of the apoproteins into VLDL, LDL, HDL fractions or a fraction with density greater than 1.18 g/ml (albumin was the major protein). Nor was there any effect by 3-deazaadenosine on the amount of phosphatidylcholine secreted into the culture medium or into VLDL or HDL. After 18 h the amount of phosphatidylethanolamine that accumulated in the cells was doubled by treatment with 3-deazaadenosine, and the amount of phosphatidylethanolamine secreted into the medium was increased by approximately 70%. It is thus apparent that the synthesis of phosphatidylcholine from ethanolamine is not required for lipoprotein secretion by rat hepatocytes.

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PubMed1985

The role of phosphatidylcholine biosynthesis in the secretion of lipoproteins from hepatocytes.

An investigation of the role of phospholipids in lipoprotein assembly and secretion is important since phospholipids, particularly phosphatidylcholine, are prominent components of all plasma lipoproteins. The fatty acid composition of phosphatidylcholine is virtually identical in human very low (VLDL), low, and high density lipoproteins, which supports the idea that phosphatidylcholine exchanges freely among plasma lipoproteins. However, the fatty acid composition of phosphatidylcholine from cultured rat hepatocytes is different from that in the secreted lipoproteins. In addition, the composition of molecular species of phosphatidylcholine is quite different in the rat liver, plasma, and red cells. Phosphatidylcholine is made in liver by two alternate pathways, by the CDP-choline pathway and by the methylation of phosphatidylethanolamine. Regulation of phosphatidylcholine biosynthesis by the CDP-choline pathway in rat liver is well established. In most instances, the rate of phosphatidylcholine synthesis is governed by the activity of CTP:phosphocholine cytidylyltransferase, which is present in the cytosol and also associated with microsomes. The cytosolic enzyme is inactive but can be reversibly translocated to the microsomes, where it is active. Translocation of this enzyme to the microsomes can be achieved either by a dephosphorylation reaction or by the presence of fatty acids in the cytosol. Once synthesized, how is phosphatidylcholine assembled into lipoprotein particles? The sequence of assembly of phospholipids into VLDL has been investigated in several studies. In a pulse-chase experiment, there was an initial labelling (within 15 min of the pulse) of phospholipids in secreted VLDL, which probably reflected the rapid movement of the phospholipids from their site of synthesis (the endoplasmic reticulum) to the Golgi. There appears to be a rapid exchange of phospholipid between the Golgi membranes and contents. There was also a delayed labelling (after 30 min) of the phospholipids and triacylglycerols (from [3H]glycerol) and the apoproteins (from [3H]leucine) in the secreted VLDL. This lag was attributed to the time taken for the nascent VLDL particles to move from the lumen of the endoplasmic reticulum to the Golgi and into the medium. Is phosphatidylcholine biosynthesis is required for lipoprotein secretion? This question was investigated in rats maintained on a choline-deficient diet for 10 days. The total amount of plasma phosphatidylcholine decreased by approximately 40% and the rats developed fatty livers.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1972