The goal of this proposal is to describe in molecular terms the structure and function of two oxidation/reduction enzymes in the secretory vesicles of neuroendocrine cells--dopamine beta-hydroxylase (DBH) and cytochrome b561. The first objective of the program is to determine the primary structure of cytochrome b561 using protein chemistry and recombinant DNA techniques. Radiolabeling of specific amino acids on native cytochrome will then be used to locate the relative membrane positions of polypeptide sections. We will also use neutron activation analysis and spectrophotometric redox titration to determine the number and type of redox sites in the cytochrome. Knowledge of the overall structure of cytochrome b561 will allow us to begin determination of the spatial organization of its redox active sites. This cytochrome appears to be an excellent model protein with which to study long range electron transfer in biological systems. The second objective is to characterize the effect of transmembrane ion gradients on the function of cytochrome b561. For this objective we will use phospholipid vesicle reconstitution, ionophores to create gradients and spectrophotometric assay of the cytochrome function. Since the cytochrome normally functions in the environment of a large transmembrane potential, these studies are necessary to understand the normal functioning of cytochrome b561. The third objective is to determine the structural differences between membranous and soluble forms of DBH. This will be done with standard protein chemistry techniques. The structural aspects of catecholamine biosynthetic enzymes which determine their respective cellular locations and subunit assembly are not understood. We are closest to this understanding with DBH, and since it appears that some of the catecholamine synthesizing enzymes are a homologous family of proteins, a detailed knowledge of the structure of DBH will help scientific progress in this field in a broad sense. The fourth objective is to kinetically characterize the allosteric regulation of DBH by ADP. Standard enzyme assay and computer analysis of the complex kinetics will be used. DBH was not previously known to be allosterically regulated. Thus, this characterization is necessary to permit an understanding of the regulation of catecholamine biosynthesis.
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