Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the stepwise transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) to the amino headgroup of PE. Successive methylation results in the formation of the two intermediates, phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N, N-dimethylethanolamine (PDME), and the final product phosphatidylcholine (PC). PE N-methyltransferase is an integral membrane protein localized primarily in the endoplasmic reticulum (microsomal fraction) of liver. PE-, PMME- and PDME-dependent PE N-methyltransferase activities were purified from Triton X-100 solubilized microsomes 429-, 1542- and 832-fold, respectively. The purified enzyme was composed of a single 18.3 kDal protein as determined by SDS-PAGE. Molecular mass analysis of purified PE N-methyltransferase (in Triton X-100 micelles) by gel filtration on Sephacryl S-300 indicated the enzyme existed as a 24.7 kDal monomer. PE N-methyltransferase catalyzed the complete conversion of PE to PC and had a pH optimum of 10 for all three steps. A Triton X-100 mixed micelle assay was developed to assay PE-, PMME- and PDME-dependent activities of both pure and microsomal PE N-methyltransferase. The AT-terminal amino acid sequence of rat liver PE N-methyltransferase and the recently cloned 23.1 kDal S. cerevisiae PEM 2 were found to be 35% homologous. Double reciprocal plots for PE N-methyltransferase at fixed Triton X-100 concentrations and increasing PE, PMME or PDME were highly cooperative. Similar cooperative effects were noted when phospholipid was fixed and Triton X-100 increased. The cooperativity could be partially abolished if a fixed mol% of nonsubstrate phospholipid such as PC was included in the assay. This would indicate that PE N-methyltransferase has specific binding requirements for a site(s) in contact with the micellar substrate. The occupation of this boundary layer by phospholipid is essential for full expression of enzyme activity. Kinetic analysis revealed that PMME and PDME methylation followed an ordered Bi-Bi mechanism. The overall mechanism involves initial binding of PE to a common site and successive methylation steps involving the binding and release of AdoMet and S-adenosyl-L-homocysteine, respectively. Cysteine residue(s) (which are rapidly oxidized in the absence of reduced thiols) are involved in the catalytic mechanism. Reverse-phase HPLC was used to fractionate the phospholipid products of PE N-methyltransferase into individual molecular species. Substrate specificity experiments on PE N-methyltransferase in vitro and in vivo revealed no selectivity for any molecular species of diacyl PE, PMME or PDME. The PE-derived PC, which is rich in 16:0-22:6, is rapidly remodeled to conform to the molecular species compositon of total hepatocyte PC in vivo . The 18.3 kDal PE N-methyltransferase was found to be a substrate for cAMP-dependent protein kinase in vitro. However, only 0.25 mol phosphorus/mol of PE Af-methyltransferase was incorporated, with no observed effect on activity. Studies on PE N-methyltransferase regulation in choline-deficient rat liver indicated that activity changes were due to elevated levels of cellular PE. Immunoblotting of choline-deficient liver microsomes or hepatocyte membranes with a anti-PE N-methyltransferase antibody revealed no alteration in enzyme mass. While more work is needed, initial indications are that hepatic PE N-methyltransferase is a constitutive enzyme regulated primarily by substrate and product levels. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Lam Mei Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgement --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- The facilitative glucose transporter family --- p.2 / Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5 / Chapter 1.3 --- Antisense strategy --- p.8 / Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9 / Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13 / Chapter 1.3.3 --- Factors influencing antisense activity --- p.16 / Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17 / Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2: --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Reagents and buffers --- p.25 / Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26 / Chapter 2.1.3 --- Culture medium --- p.28 / Chapter 2.1.4 --- Chemicals --- p.29 / Chapter 2.1.5 --- Culture of cells --- p.31 / Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31 / Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32 / Chapter 2.1.6 --- Animal Studies --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- In vitro studies --- p.34 / Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34 / Chapter 2.2.1.2 --- Transfection --- p.35 / Chapter 2.2.1.3 --- MTT assay --- p.36 / Chapter 2.2.1.4 --- Flow cytometry --- p.37 / Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45 / Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46 / Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47 / Chapter 2.2.1.8 --- Western blot analysis --- p.50 / Chapter 2.2.2 --- In vivo studies --- p.55 / Chapter 2.2.2.1 --- Animal studies --- p.55 / Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58 / Chapter (ii) --- Creatine kinase (CK) assay --- p.60 / Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62 / Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64 / Chapter Chapter 3: --- Results --- p.67 / Chapter 3.1 --- In vitro studies --- p.68 / Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68 / Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77 / Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82 / Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87 / Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96 / Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100 / Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108 / Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117 / Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125 / Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131 / Chapter 3.2 --- In vivo studies --- p.135 / Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135 / Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139 / Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139 / Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147 / Chapter Chapter 4: --- Discussion --- p.151 / Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152 / Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154 / Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155 / Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158 / Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166 / Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167 / Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168 / Chapter 4.3 --- Conclusion --- p.169 / Bibliography --- p.172