Our competing continuation application on the biochemistry of the exocrine pancreas is concerned with: 1/ NEW MAMMALIAN GUT-BRAIN PEPTIDES OF THE VIP/PHI/SECRETIN FAMILY STIMULATING THE EXOCRINE PANCREAS: we will isolate a) PHI variants and b) mammalian helodermin(s). Mammalian helodermins are parent peptide(s) of lizard helodermin, a pentatriacontapeptide-amide we isolated 2 years ago from the sublingual venom of the Helodermae lizard Gila Monster. We will purify these peptides from: a) """"""""post-secretinic"""""""" fractions from pig small intestine provided by Dr. Mutt); b) selected human endocrine tumors; and c) brain, gut, and stomach from rat. Once the amino acid sequence established and synthetic equivalents made available, their biochemical properties on the rat pancreas will be defined in terms of cyclic AMP levels, amylase secretion, binding to receptors, etc; 2/ SECRETIN, VIP and CCK RECEPTORS. To pursue the molecular characterization of secretin-preferring and VIP-preferring receptors, pancreatic membranes from rat (and accessorily from guinea pig and man) will be solubilized after crosslinking with new radioiodinated ligands. We hope to further purify CCK receptors by immunoaffinitychromatography with anti CCK receptor monoclonal antibodies or antibodies from the serum of patients with pernicious anaemia. Utilizing pancreatic receptors as a tool, VIP and secretin analogs specifically endowed with superagonist or antagonist properties and highly specific for each class of receptor will be taylored; 3/ MUSCARINIC RECEPTORS: the study of their effector coupling through regulatory protein(s) will help to delineate properties typical of the B subclass of M2 receptors (with intermediate affinity for pirenzepine); 4/ GUANINE NUCLEOTIDE REGULATORY PROTEINS Ns, Ni and Nx. We will try to identify Nx, a protein distinct from Ni and Ns that might allow the coupling of high affinity pancreatic CCK receptors and muscarinic receptors with phospholipase C; 5/ THE INTERMEDIARY METABOLISM OF THE EXOCRINE PANCREAS. We will focus our attention on 3 topics: 1) endogenous phospholipase A2 (distinct from secretory prophospholipase A2) and its possible role in stimulus-secretion coupling and as an agent of cell damage, using as standard of comparison a new type of phospholipase A2 we recently isolated from the venom of Heloderma suspectum; 2) the structure of a small regulatory phosphoprotein from pancreatic endoplasmic reticulum (Mr 21 kDa); and 3) the effect of """"""""invasive adenylate cyclase"""""""", an enzyme we wish to isolate from Bordetella pertussis. This bacterial enzyme, once in acinar cells, could be remarquably regulated by intracellular calmodulin and calcium fluxes.
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