Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [160]-167).

No description available.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: School of Medicine. Bibliography: leaves 140-149. Also available online.

Several model systems were investigated to elucidate the mechanisms by which rat liver phosphatidylcholine synthesis is controlled. CTP: phosphocholine cytidylyltransferase was clearly the key regulatory enzyme for phosphatidylcholine formation from choline. This ambiquitous enzyme was detected in both the cytosolic and microsomal fractions of rat liver, although the majority of the cytidylyltransferase occurred in the soluble fraction. The distribution of cytidylyltransferase between these fractions was altered when the rate of phosphatidylcholine synthesis was perturbed. Translocation of cytidylyltransferase was observed in rat liver during early development, with starvation and during a diurnal rhythm. A redistribution of cytidylyltransferase was also detected in isolated hepatocytes which were treated with glucagon, cAMP analogues or fatty acids bound to albumin. The rate of phosphatidylcholine synthesis was found to reflect the amount of microsomal cytidylyltransferase activity. The inhibition of phosphatidylcholine synthesis by glucagon or cAMP analogues was likely due to phosphorylation and inhibition of the cytidylyltransferase. Several lines of evidence indicated that the cytidylyltransferase in fresh rat liver cytosol was probably phosphorylated and activated upon dephosphorylation by endogenous phosphoprotein phosphatases or alkaline phosphatase from hog intestine. Although the phosphorylation of cytidylyltransferase was apparently kinetically "silent", dephosphorylation resulted in an increased affinity of the enzyme for membranes. Fatty acids stimulated de novo phosphatidylcholine synthesis by acceleration of the cytidylyl-transferase-catalyzed reaction. Fatty acids and their CoA derivatives were shown to stimulate the cytosolic cytidylyltransferase activity. However, these compounds failed to activate partially purified cytidylyltransferase appreciably. Apparently, fatty acids, like dephosphorylation, enhanced the tenacity of cytidylyltransferase for membranes. Upon binding to membranes, cytidylyltransferase activity could be elevated up to 45-fold, and the affinity of the enzyme for the substrate, CTP, was increased 20-fold. The influence of glucagon, cAMP analogues and fatty acids on the synthesis of phosphatidylcholine by successive N-methylation was also examined in isolated rat hepatocytes. Glucagon and cAMP analogues inhibited the methylation pathway in these cells, but the activity of microsomal phosphatidylethanolamine methyltransferase was elevated. Fatty acids also reduced the formation of phosphatidylcholine from phosphatidylethanolamine. Fatty acids and their CoA derivatives directly inhibited the phosphatidylethanolamine methyltransferase in rat liver microsomes. The coordinate control of hepatic phosphatidylcholine synthesis by cAMP and fatty acids may be important during starvation when the intracellular levels of these compounds are increased. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Two kinds of affinity chromatography, CDP-choline- and CTP-Sepharose 4B, were investigated for purification of the cytosolic CTP:phosphocholine cytidylyltransferase from rat liver. The enzyme did not show strong affinity for the CDP-choline Sepharose resin, but bound to the CTP-Sepharose column in the presence of 14 mM magnesium acetate. The combination of CTP affinity chromatography with ion-exchange techniques provided about 70-fold purification of the cytosolic enzyme with a specific activity of about 90 units per milligram protein. The influence of diphenylsulfone compounds on the synthesis of phosphatidylcholine by the CDP-choline pathway was examined in isolated rat hepatocytes and HeLa cells. The administration of the sulfones (100 ug/ml), except dapsone, to HeLa cells inhibited the total [methyl-³H]choline incorporation into the cells, but did not change the rate of conversion of choline to phosphatidylcholine. The addition of the sulfones (100 ug/ml) to rat hepatocytes did not inhibit the biosynthesis of phosphatidylcholine and choline metabolism. The effect of vasopressin on the distribution of cytidylyl-transferase between cytosol and microsomes in rat hepatocytes was also investigated. The digitonin-mediated release of cytosolic cytidylyltransferase was reduced from the cells treated with vasopressin (5-20 nM) , while the enhanced rate of incorporation of [methyl-³H]choline into phosphatidylcholine was not observed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

The last step in sphingomyelin's de novo biosyn-thetic pathway was investigated in Baby Hamster Kidney (BHK-21F) cells in tissue culture. Three types of pulse-chase experiments were done to try to identify the precursor for sphingomyelin's phosphocholine moiety. First, [Me- ³H]-choline was used to monitor the movement of the choline moiety in all the possible phosphocholine donors: ie. phosphocholine, CDP-choline, phosphatidylcholine, lysophospha-tidylcholine, and glycerophosphocholine. Radioactivity was observed in phosphocholine before appearing in phosphatidylcholine, glycerophosphocholine, and sphingomyelin. Specific radioactivities of phosphatidylcholine and sphingomyelin revealed a peculiar pattern, if representative of a precursor-product relationship between these two phospholipids. Their specific radioactivities became equal at 22 hours of chase and remained quite similar for the next 24 hours. The other two types of pulse-chase experiments both utilized prelabeled BHK phosphatidyl[Me- ³H]choline in phospholipid vesicles as their 'pulse' source. Phospholipid Exchange Protein(PLEP)-mediated exchange and polyethylene glycol/phytohemagglutinin (PEG/PHA)-mediated fusion between phospholipid vesicles and BHK cells were used to introduce the labeled phosphatidylcholine. In addition, in the 'fusion' experiments, other labeled compounds (glycerophosphocholine and sphingomyelin) were substituted for labeled phosphatidylcholine. PLEP- mediated exchange of labeled phosphatidylcholine did not result in enough transfer of radioactivity into the cell to adequately monitor individual cell phospholipids or any transfer of label - ie. from phosphatidylcholine to sphingomyelin. However, PEG/PHA-mediated fusion of vesicles and cells did result in enough radioactivity showing up in the cells. When labeled phosphatidylcholine was used, the radioactivity ratio between it and sphingomyelin averaged around 16, depending on the length of the chase (2-52 hours)m The use of either labeled glycerophosphocholine or sphingomyelin resulted in a ratio of about 1.8. One 'cold-trap1 experiment was done by including a large amount of unlabeled glycerophosphocholine with labeled phosphatidylcholine in the vesicle preparation. The resultant radioactivity in sphingomyelin was 50% less than previously, but phosphatidylcholine's had remained the same. The evidence seems to indicate a reversible precursor-product relationship between phosphatidylcholine and sphingomyelin, but does not clearly show whether or not any other intermediate (such as glycerophosphocholine) is also involved. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Københavns Universitet.