The overall goals of the research are to understand the factors regulating the content of the polyamines spermidine and spermine in mammalian cells and to use this knowledge to manipulate cellular polyamine levels and evaluate the role of the polyamines in normal and neoplastic growth. The proposed investigations will focus predominantly on S-adenosylmethionine decarboxylase (AdoMetDC) although some studies on the regulation of ornithine decarboxylase (ODC) by polyamines will also be carried out. Previous work supported by this grant has led to the production of monospecific antibodies to these enzymes and of cDNA clones for AdoMetDC. Radioimmunoassays for these proteins and means of labelling their active sites have also been developed. These tools and reagents will be used for the proposed studies.
The specific aims of the research proposal are: (1) To obtain the complete cDNA sequence corresponding to rat AdoMetDC mRNA and to derive amino acid sequence of AdoMetDC and its precursor. (2) To investigate the biosynthesis and processing of the AdoMetDC precursor using antisera to AdoMetDC to precipitate the relevant proteins from a reticulocyte lysate protein synthesis system supplemented with the mRNA. (3) To investigate the regulation of ODC and AdoMetDC protein levels and synthesis rates by polyamines. (4) To continue the study of the effects of polyamines on the translation of the mRNA for AdoMetDC and ODC which we have found to be much more sensitive to inhibition by polyamines than other mammalian mRNAs. (5) To obtain CHO cell mutants lacking AdoMetDC and use these to study the regulation of the synthesis of the enzyme by transfection of constructs containing the AdoMetDC gene. (6) To obtain genomic clones for AdoMetDC and use them to study the regulation of enzyme synthesis. (7) To isolate and characterize cDNA clones for AdoMetDC from human and trypanosome sources. Our previous work has shown that AdoMetDC from rat prostate contains a covalently bound pyruvate prosthetic group and that the enzyme is synthesized as a precursor of Mr 37,000 which is converted to the enzyme sub-unit of Mr 32,000 in a reaction which presumably generates this prosthetic group. The proposed work will delineate the biochemical mechanism underlying the synthesis of this enzyme and will provide new information on the regulation of its biosynthesis.
Showing the most recent 10 out of 106 publications
