Studies will be carried out on ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are the key regulatory enzymes in the synthesis of polyamines in mammalian cells and are highly inducible in response to growth-promoting stimuli. The distribution, regulation of the activity, and effects of inhibition of these enzymes by specific inhibitors will be investigated. Methods of purifying both enzymes to homogeneity from mammalian tissues have been developed, and antibodies to the purified proteins produced. These antibodies were used to set up RIA procedures that were used to quantitate the amount of protein and to isolate cDNA probes for their mRNAs. Also, it has been shown that ornithine decarboxylase can be labeled specifically in vitro and in vivo by the covalent attachment of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor. The binding of DFMO will be used to quantitate the amount of active ornithine decarboxylase present in tissue extracts, to study the biodegradation of ODC, which has a very short half-life, and to provide a method for autoradiographic localization of the enzyme in tissue sections. A mutant HTC cell line in which the half-life of ODC is greatly increased, but the turnover of other proteins is not altered, will be used to define the factors responsible for the degradation of ODC. Peptide-mapping techniques will be used to study the structure of ODC, the binding of DFMO, and the possible existence of multiple forms of the enzyme. Similar analysis will be carried out on S-adenosylmethionine decarboxylase, which our previous studies have shown exists in two different forms that differ in their sensitivity to the antitumor drug, methylglyoxal bis(guanylhydrazone) (MGBG). The nature of the difference between these forms and their distribution in normal and neoplastic tissues will be investigated. The mechanism of biodegradation of S-adenosylmethionine decarboxylase, which also turns over rapidly but is stabilized by MGBG, and of the regulation of this enzyme by spermidine will be studied. The inhibition by DFMO and MGBG of polyamine biosynthesis via these decarboxylases may provide a means of inhibiting tumor growth, and these studies will provide information on these enzymes needed to evaluate and optimize such effects. (B)
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