1). The major products from the non-ACTH/non-LPH region of the ACTH/endorphin common precursor in rat anterior and intermediate pituitary and mouse corticotropic tumor cells (AtT-20) will be defined by characterization of product peptides and by biosynthetic labeling experiments. An immunoassay for joining peptide will be developed and the Alpha-amidation state of joining peptide will be determined. The extent of glycosylation within the non-ACTH/non-LPH region of the precursor and the effect of glycosylation on biosynthetic proteolysis will be studied. The time course of post-translational processing and the possibility of destructive proteolysis of selected peptide products will be investigated. 2). Secretory granule-associated peptide Alpha-amidation activity will be purified from bovine intermediate pituitary and AtT-20 cells and characterized by protein chemical and enzymologic approaches. The stoichiometry of the Alpha-amidation reaction will be established, now that the basic scheme of the reaction is known. Antibodies to Alpha-amidation activity will be produced and used for immunoassays and immunostaining. Kinetic studies will begin to define the order of the reaction. 3). The ability of dietary or genetic alterations in the availability of copper and ascorbate to affect peptide Alpha-amidation in the pituitary and in other tissues producing Alpha-amidated peptides will be determined. The amidation state of Alpha MSH and joining peptide will be determined directly and a chemical assay for amino acid Alpha-amides will provide a general assessment of functional Alpha-amidation activity in vivo. The sensitivity of Alpha-amidation to copper and ascorbate deficiency will be compared to the sensitivity of other copper- and ascorbate-dependent enzymes. AtT-20 cells will serve as a tissue culture model of the effects of copper, ascorbate, and other factors on Alpha-amidation; uptake and packaging of copper and ascorbate into cells and secretory granules will be studied. 4). Appropriate synthetic peptides will be used to identify and purify two biosynthetic proteases that seem to be unique to intermediate as opposed to anterior pituitary ACTH/endorphin cells. Purified secretory granules will be used as starting material and lysosomal contamination will be closely monitored. Products will be rigorously identified by high performance liquid chromatography. Purified biosynthetic proteases will be used for studies of enzyme specificity, tissue localization, and biological regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK032949-09
Application #
3483638
Study Section
Endocrinology Study Section (END)
Project Start
1983-04-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
9
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Maheshwari, Sweta; Shimokawa, Chizu; Rudzka, Katarzyna et al. (2018) Effects of copper occupancy on the conformational landscape of peptidylglycine ?-hydroxylating monooxygenase. Commun Biol 1:74
Kumar, Dhivya; Strenkert, Daniela; Patel-King, Ramila S et al. (2017) A bioactive peptide amidating enzyme is required for ciliogenesis. Elife 6:
Bäck, Nils; Kanerva, Kristiina; Kurutihalli, Vishwanatha et al. (2017) The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together. Eur J Cell Biol 96:407-417
Vishwanatha, Kurutihalli S; Bäck, Nils; Lam, TuKiet T et al. (2016) O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking. J Biol Chem 291:9835-50
Kumar, Dhivya; Mains, Richard E; Eipper, Betty A (2016) 60 YEARS OF POMC: From POMC and ?-MSH to PAM, molecular oxygen, copper, and vitamin C. J Mol Endocrinol 56:T63-76
Bonnemaison, Mathilde L; Duffy, Megan E; Mains, Richard E et al. (2016) Copper, zinc and calcium: imaging and quantification in anterior pituitary secretory granules. Metallomics 8:1012-22
Kumar, Dhivya; Blaby-Haas, Crysten E; Merchant, Sabeeha S et al. (2016) Early eukaryotic origins for cilia-associated bioactive peptide-amidating activity. J Cell Sci 129:943-56
Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E et al. (2015) Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine ?-Amidating Monooxygenase, a Luminal Cuproenzyme. J Biol Chem 290:21264-79
Simpson, Peter D; Eipper, Betty A; Katz, Maximiliano J et al. (2015) Striking Oxygen Sensitivity of the Peptidylglycine ?-Amidating Monooxygenase (PAM) in Neuroendocrine Cells. J Biol Chem 290:24891-901
Gaier, E D; Rodriguiz, R M; Zhou, J et al. (2014) In vivo and in vitro analyses of amygdalar function reveal a role for copper. J Neurophysiol 111:1927-39

Showing the most recent 10 out of 33 publications