The long term goal of this project is to understand the biosynthesis of cytokinins, a class of plant growth hormone. The project focuses on the first enzyme in the cytokinin biosynthetic pathway: 5'-AMP-isopentenyltransferase (ITPase). This enzyme has been detected in cultured cytokinin-habituated tobacco tissue that will serve as a source of this enzyme for further purification using conventional methods (batch, columns, HPLC). Antibodies against this partially purified enzyme will be produced and purified by passing the antiserum through a Sepharose-4B column coupled to soluble proteins obtained from the cytokinin-dependent tobacco tissue which lacks IPTase activity. The purified antibody preparation will be used for immunoaffinity chromatography to further purify the enzyme. The kinetic properties of the enzyme will be studied. The enzyme ITPase is the first enzyme in the cytokinin biosynthetic pathway. It is likely to catalyze the rate-limiting step in cytokinin biosynthesis. An enzyme with similar catalytic properties but not genetically homologous to the plant enzyme has been isolated from plants transformed by Agrobacterium tumefaciens. Unfortunately the lack of homology between the Agrobacterium IPTase gene and plant sequences means that the gene for the plant enzyme must be obtained by conventional means. This project is the first step toward isolating the gene for the IPTase of plants. Understanding the regulation of this gene in plants is important to our overall understanding of growth and development in plants.
