Molecular characterization and identification of antigen 32-5B6 as enzyme S-Adenosyl-L-Homocystein-Hydrolase from Xenopu

Diplomarbeit aus dem Jahr 1996 im Fachbereich Biologie - Genetik / Gentechnologie, Note: keine Angabe, Eberhard-Karls-Universität Tübingen (Max-Planck-Institut für Entwicklungsbiologie, Tübingen), Veranstaltung: -, Sprache: Deutsch, Abstract: Molecular characterization and identification of antigen 32-5B6 as enzyme S-Adenosyl-L-Homocystein-Hydrolase from Xenopus laevis oocyte nuclei Claudia MohlDiploma thesis in Biochemistry, Eberhard-Karls-University, Tübingen, 1996 and Max-Planck-Institut for Development Biology, Tübingen(Title and Abstract in english; Original work in german)Abstract:The aim of my diploma thesis work (1996) was molecular characterization and identification of late migrating antigen 32-5B6 on a molecular level. Antigen 32-5B6 is a protein that is distributed in cytoplasm during Blastula stage and transported into oocyte cell nuclei at Gastrula stage 12 during embryonic development of Xenopus laevis (Dreyer et al. 1982; 1983). To isolate cDNA sequences that encode the late migrating antigen 32-5B6, I screened, isolated and sequenced five cDNA clones from placques with positive antibody reaction from a Xenopus laevis ovar lambda zap II cDNA expression library. Sequence analysis showed that two cDNA clones encode the enzyme S-Adenosyl-Homocystein-L-Hydrolase (clone 10) from Xenopus laevis (Seery et al. 1994) and an isoform of this enzyme (clone 8). Molecular weight and IEP of S-Adenosyl-Homocystein-L-Hydrolase are nearly identical with those of antigen 32-5B6. To get further evidence in regard to sequence of antigen 32-5B6, proteins were isolated from original Xenopus laevis oocyte nuclei for protein microsequencing. For this reason I established a new protein purification strategy purifying proteins from original oocyte nuclei of Xenopus laevis proteom by means of anion-exchange chromatography and 2-dimensional gel electrophoresis. By means of western blot analysis I could detect two enzyme isoforms those IEPs lie in range between pH 6.02 - 6.2 and