Mitchell, John L. A.
M.S. (Master of Science)
Department of Biological Sciences
Ornithine decarboxylase; Enzymes; Cytochemistry; Mammals--Cytology
Ornithine decarboxylase (ODC; E.C.22.214.171.124) catalyzes the first and rate limiting step in the polyamine biosynthetic pathway. Its activity responds to a variety of hormones and other stimuli that control cell growth and differentiation. Although much work has been done on the isolation of multiple ODC forms from rat tissue and HTC cells in culture, the use of anion exchange columns to distinguish the forms of ODC has not, as yet, been applied to mouse kidney, a very rich source of ODC protein. In this study, crude and highly purified (3000-fold) ODC isolated from mouse kidney could be separated into three peak areas. The first and second peak areas off the Mono 0 anion exchange column, ODC I and II respectively, rechromatographed as distinct ODC isoforms. The combined fractions beyond the peak II area eluted as both peaks I and II. Thus it appears that enzyme activity beyond peak II represents dimeric combinations of monomer forms I and II. The distinct charge difference between ODC I and II is still observed in partially and highly purified mouse kidney ODC labeled with ³H-DFMO and denatured with 8.0 M urea. In an effort to determine whether or not the two charge forms separated by anion exchange chromatography correlated with the two isoforms observed on 2-dimensional gels, the two major forms labeled with ³H-DFMO, separated on the Mono Q ion- exchange column, exhibited distinct isoelectric points which directly correlate with the isoelectric points others have observed using 2-dimensional gel electrophoresis. To facilitate this study of the mouse kidney ODC forms and the rapid degradation of this enzyme, an antibody was prepared against mouse kidney ODC. The purification of this enzyme included centrifugation, ammonium sulfate fractionation, DEAE C1-6B Sepharose anion exchange, and pyridoxamine phosphate affinity chromatography.
Rynning, Michael Douglas, "Separation and identification of isoforms of mammalian ornithine decarboxylase" (1987). Graduate Research Theses & Dissertations. 4882.
ix, 91 pages
Northern Illinois University
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