The primary objectives of the work proposed are to provide specific information about the metabolic regulation, molecular biology and relatedness (homology) of the key isofunctional enzyme NAD kinase (E.C.2.7.1.23) from Escherichia coli, Bacillus subtilis and other bacteria, NAD which form NADP from NAD and ATP. Previous studies from this laboratory have shown that the primary function of the NAd cycle is to maintain relatively constant levels of NAD and NADP in bacteria, employing a de novo biosynthetic pathway, exogenous precursors and endogenous products resulting from the degradation of NAD. Thus NAD kinase, an allosteric enzyme in E. coli and B. subtilis, plays an essential role in the NAD cycle as well as, in support of biosynthesis. More comprehensive studies of the metabolic regulation of the NAD kinase from these two mechanisms and B. licheniformis is planned, seeking to identify regulatory metabolites originating in the major amphibolic and anaplerotic pathways. The NAD kinase genes from E. coli and B. subtilis will be cloned from existing and proven genomic libraries using established procedures in order to 1) amplify the genes and gene products in transformed host cells; 2) map the genes in the relevant chromosomes since their loci are unknown; 3) use the NAD kinase genes isolated as probes in evaluating DNA homology in related and unrelated bacteria from various genera and species. The polyclonal antibodies raised against the NAD kinase from E. coli and B. subtilis and used in the cloning and subcloning of the NAD kinase genes, will also be used in """"""""relatedness"""""""" studies with a variety of bacteria, employing the Ouchterlony double- immunodiffusion technique. The results obtained from the immunodiffusion and DNA homology studies will help us in the selection of representative organisms in which to determine the regulatory characteristics of the NAD kinase. These results will help to achieve another important aim, i.e. to define the different types or classes of metabolic regulation of the isofunctional NAD kinase which exist in an array of important groups of bacteria. The information obtained may also be useful to those concerned with the evolution of genes and gene products and bacterial taxonomy.
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