Conventional column chromatographic procedures enabled a 1000 fold purification of a novel porcine pituitary proteinase, called IRCM-Serine Protease #1. The cleavage specificity of this enzyme towards small fluorogenic substrates and the native POMC - related polypeptide hormones ACTH 1-39, Beta-LPH 1-89 and N-terminal 1-76 and its subcellular distribution suggest that the enzyme may participate in pro-hormone processing in vivo. It is proposed to test this hypothesis, by obtaining enough enzyme for primary sequence determination in order to obtain specific antisera for immunohistochemical co-localization studies with POMC and other pro-hormones. Due to the low levels of enzyme (12 Mug/50 g wet weight) and the need of another 500-1000 fold purification, novel affinity column procedures using peptidyl-arginal columns are being developed. The latter, will be of general use in the purification of """"""""trypsinlike"""""""" Serine and Cysteinyl proteases. Furthermore, the direct microsequencing of electroblotted proteins from SDS gels should provide the necessary sensitivity at the 1-10 pmole level. Although initial experiments are being performed with proteinases purified from porcine pituitaries, we have the unique opportunity to have access during the term of this grant to nearly 15,000 frozen human pituitaries. These will be used to extend our knowledge to """"""""human maturases"""""""" and will constitute the major aim of this application. In addition the sequence information should allow the molecular cloning of this enzyme in order to obtain the cDNA sequence and the genomic organization of this protease in human. The antibodies and cDNA probes will be used to follow the variation in the levels of this enzyme during various physiological stimuli, allowing a further definition of the factors involved in its physiological regulation. The purification can be adapted for the isolation and characterization of the homologous enzyme from frozen human pituitaries and other tissues. Nucleotide probe screening can be done in human cDNA libraries. Additionally, we are currently characterizing other proteases isolated from human pituitaries in order to assess their possible involvement in pro-hormone maturation. The characterization of the enzyme system(s) involved in the conversion of precursor proteins into their end-products is a key step for the understanding and future research endeavors in the expanding field of neuropeptides, particularly since it will apply to human glands.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS016315-07
Application #
3396806
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1979-12-01
Project End
1989-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Clinical Research Institute of Montreal
Department
Type
DUNS #
City
Montreal
State
QC
Country
Canada
Zip Code
H2 1R7
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Cromlish, J A; Seidah, N G; Marcinkiewicz, M et al. (1987) Human pituitary tryptase: molecular forms, NH2-terminal sequence, immunocytochemical localization, and specificity with prohormone and fluorogenic substrates. J Biol Chem 262:1363-73
Seidah, N G; Donohue-Rolfe, A; Lazure, C et al. (1986) Complete amino acid sequence of Shigella toxin B-chain. A novel polypeptide containing 69 amino acids and one disulfide bridge. J Biol Chem 261:13928-31
Bennett, H P; Seidah, N G; Benjannet, S et al. (1986) Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1-76). Int J Pept Protein Res 27:306-13
Cromlish, J A; Seidah, N G; Chretien, M (1986) A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates. J Biol Chem 261:10850-8
Burbach, J P; Seidah, N G; Chretien, M (1986) Isolation and primary structure of novel neurointermediate pituitary peptides derived from the C-terminal of the rat vasopressin-neurophysin precursor (propressophysin). Eur J Biochem 156:137-42
Cromlish, J A; Seidah, N G; Chretien, M (1986) Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles. J Biol Chem 261:10859-70
Gaspar, L; Chan, J S; Seidah, N G et al. (1985) Characterization of peptides derived from pro-opiomelanocortin in the biological fluids of a patient with Nelson's syndrome. Clin Invest Med 8:167-75
Marcinkiewicz, M; Benjannet, S; Seidah, N G et al. (1985) Immunocytochemical localization of a novel pituitary protein (7B2) within the rat brain and hypophysis. J Histochem Cytochem 33:1219-26
Marcinkiewicz, M; Benjannet, S; Sikstrom, R A et al. (1985) Immunoreactivity of vasopressin and a novel pituitary protein '7B2' in Long-Evans and Brattleboro rat hypothalamus and hypophysis. Neurosci Lett 60:7-12