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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9218983 Purification, characterization, and inhibition of S-adenosyl-X-methionine decarboxylase (E.C. 4.1.1.50) from Acanthamoeba castellanni N eff Hugo, Eric R., Ph.D. The Ohio State University, 1992 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 Purification, Characterization, and Inhibition of S-Adenosyl-L-Methionine Decarboxylase (E.C. 4.1.1.50) from Acanthamoeba castellanii Neff DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University by Eric R. Hugo, B.S., M.S. * * * * * The Ohio State University 1992 Dissertation Committee Approved by Thomas J. Byers, Committee Chair Berl O. Oakley Donald H. Dean Adviser Roy H. Tassava Department of Molecular Genetics To Vickie ACKNOWLEGEMENTS I would like to thank Drs. Thomas and Sandra Byers for their gracious help during my tenure in Columbus. I would also like to thank Dr. Wayne Parrish for support during part of this study as well as for many helpful discussions. I am greatly appreciative of the all of the assistance Ms. Rebbecca Gast provided during my stay in Dr. Byers laboratory. Finally, I thank my wife Vickie for her love, kindness, and patience during the many long hours I spent working in the laboratory. VITA November 27,1961 ...................................................Born Ft. Thomas, Ky. May, 1983 ................................................................ B.S. Northern Kentucky University Highland Heights, Ky. 1983-1986 ................................................................ Graduate Teaching Associate Dept, of Microbiology The Ohio State University Columbus, OH December, 1986 .......................................................M.S. The Ohio State University Columbus, OH 1986-1990 ............................................................... Graduate Teaching and Research Associate, Dept, of Molecular Genetics, The Ohio State Univesity, Columbus, OH 1990-199 1................................................................. Instructor, Denison University Granville, OH 1991-presen t ............................................................. Research Associate, DepL of Biochemistry and Molecular Biophysics, Washington University School of Medicine St. Louis, MO PUBLICATIONS Abstracts: Byers, T.J. and E.R. Hugo. 1990. S-adenosylraethionine decarboxylase: a possible role in the encystment of Acanthamoeba castellanii. Invest. Opthal. Vis. Sci. Abstract 2066 31:420. Hugo, E.R. and T.J. Byers. 1989. Purification and characterization of S-adenosyl-L-methionine decarboxylase from Acanthamoeba castellanii. J. Ceh Biol. Abstract 1403 109:255a. Hugo, E.R. and T.J. Byers. 1989. S-adenosylmethionine decarboxylase of Acanthamoeba castellanii (Neff strain). J. Protozool Abstract 198 37(4) :42A Papers and Proceedings Byers, T.J., B.G. Kim, L.E. King, and E.R. Hugo. 1991. Molecular aspects of the cell cycle and encystment in Acanthamoeba. Rev. Infect. Dis.13(Suppl 5):S378-84. Hugo, E.R., W.R. McLaughlin, K. Oh, and O.H. Tuovinen. 1991. Quantitative enumeration of Acanthamoeba for evaluation of cyst inactivation in contact lens care solutions. Invest. Vis. Ophthalmol. Res.32(3):655-57. Byers, T.J., E.R. Hugo, and V. Stewart. 1990. Genes of Acanthamoeba: DNA, RNA and protein sequences (a review), J. Protozool. 37(4):17S-25S. FIELDS OF STUDY Major Field: Molecular Biology v ABBREVIATIONS apparent Km ab: antibody agm: agmatine MTA: methylthioadenosine Ber: berenil NEM: Neffs Encystment medium BLOTTO Bovine-lacto transfer Norspd: norspermidine optimizer CBB: Coumassie Brilliant Blue OGMA: optimal growth medium A Dap: 1,3-diaminopropane Ora: ornithine dcSAM: decarboxylated SAM ODC: ornithine decarboxylase DFMO: a-difluoromethylomithine Pi: inorganic phosphate DGM-11: defined growth medium PA: polyamine DTT: dithiothreatol Pent: pentamidine EB: ethidium bromide Put: putrescine EEA: encystment enhancing act. Pro: propamidine HPLC: high performance liquid SAMDC: S-adenosylmethionine chromatography decarboxylase HS: hydroxystilbamidine SAM: S-adenosylmethionine kDa: kilodalton TPCK: tosylphenylalanyl chloromethyl ketone Kl: inhibition constant velocity TABLE OF CONTENTS ACKNOWLEGEMENTS ............................................................................................. iii VTTA .................................................................. ............................................................iv LIST OF FIGURES___________________________________________________ xi LIST OF TABLES_____________________________________________________xiv Introduction. ---------------------------------------------------------------------------------------- 1 1. Literature Review-------------------- --------------------------------------------------------- 3 l.L Polyamines _________________________________________________ 3 1.1.1 Polyamine function ___________________________________ 3 1.1.2 Polyamine synthesis ...................................................................... 7 1.1.3 Regulation of polyamine biosynthesis ............................................12 1.1.4 Polyamine function and synthesis as targets of chemotherapy -------- --------------------------------------------14 1.2 Acanthamoeba ______________________________________________ 19 1.2.1 General characteristics..................................................................19 1.2.2 Developmental biology .. ......................................................... 21 1.2.3 Polyamine metabolism ................................................................-2 4 1.2.4 Molecular biology ............................................................ 25 1.2.5 Pathogenicity ..................................................................................26 2. Materials and Methods ...............................................................................................31 2.1 Cell culture and growth ................................................................................31 2.1.1 Cell lines ........................................................................................ 31 2.1.2 Media..............................................................................................32 2.1.3 Cell growth ..................................................................................... 32 2.1.4 Effect of diamidines on growth ......................................................33 2.2 Enzyme purification ...................................................................................... 34 2.2.1 Homogenization and extract preparation .......................................34 2.2.2 Precipitation ................................................................................... 35 2.2.3 Hydrophobic interaction chromatography .....................................35 2.2.4 DEAE anion exchange chromatography .......................................36 2.2.5 Gel filtration chromatography ........................................................37 2.3 Electrophoresis ..............................................................................................37 2.3.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 37 2.3.1 Agarose isoelectric focusing gel electrophoresis .......................... 38 2.4 SAMDC assay...............................................................................................40 2.4.1 Quantitative microassay .................................................................40 2.4.2 Qualitative microtiter plate assay .................................................. 43 2.4.3 Determination of pH optima ..........................................................44 2.4.4 Kinetic analysis ..............................................................................44 2.5 Physical structure analysis .............................................................................44 2.5.1 Determination of a-subunit size ..................................................... 44 2.5.2 N-terminal amino acid sequence determination ............................ 46 2.5.3 Tryptic peptide