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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027695-08
Application #
3274919
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1980-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Georgetown University
Department
Type
School of Medicine & Dentistry
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Gibson, K R; Vanek, P G; Kaloss, W D et al. (1993) Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide. J Biol Chem 268:9490-5
Fleming, P J; Kent, U M (1991) Cytochrome b561, ascorbic acid, and transmembrane electron transfer. Am J Clin Nutr 54:1173S-1178S
Oyarce, A M; Fleming, P J (1991) Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells. Arch Biochem Biophys 290:503-10
Kent, U M; Fleming, P J (1990) Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed. J Biol Chem 265:16422-7
Taylor, C S; Kent, U M; Fleming, P J (1989) The membrane-binding segment of dopamine beta-hydroxylase is not an uncleaved signal sequence. J Biol Chem 264:14-6
Oyarce, A M; Fleming, P J (1989) Deglycosylated membranous and soluble dopamine beta-hydroxylase have identical apparent molecular weights. J Mol Neurosci 1:171-5
Srivastava, M; Fleming, P J; Pollard, H B et al. (1989) Cloning and sequencing of the human nucleolin cDNA. FEBS Lett 250:99-105
Kent, U M; Fleming, P J (1987) Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems. J Biol Chem 262:8174-8
Dhawan, S; Duong, L T; Ornberg, R L et al. (1987) Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase. J Biol Chem 262:1869-75
Fleming, P J; Kent, U M (1987) Secretory vesicle cytochrome b561: a transmembrane electron transporter. Ann N Y Acad Sci 493:101-7

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