title: Acidic pH- and metal ion (Zn++ or Mn++)-dependent proteolysis of 140 kDa atrial natriuretic factor receptor in bovine adrenal cortex plasma membranes: evidence for membrane-bound acidic metalloendopeptidase.
abstract: Incubation of the adrenal membranes at pH 3.5-5.6 resulted in apparent proteolysis of 140 kDa protein to yield a 70 kDa polypeptide containing an ANF-binding site, which could be photoaffinity labeled by [125I]4-azidobenzoyl monoiodo ANF-(4-28). This 70 kDa fragment was found to be disulfide-linked to the remaining segment(s) of the molecule, giving a total apparent Mr of 140,000 when not reduced. The acidic pH-dependent proteolysis was rapid even at 0 degree C, suggesting close association of an endopeptidase with ANF receptor. The proteolysis was inhibited by EDTA, but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide or pepstatin, indicating that the enzyme is a metalloendopeptidase. The inhibition was reversed by ZnCl2 or MnCl2, but not CaCl2 or MgCl2. The adrenal membranes contained guanylate cyclase activity of 1.1 nmol/min/mg protein using Mn-GTP as a substrate, which could be stimulated by 0.1 microM ANF to 2.7 nmol/min/mg. The membranes showed high affinity to ANF-(1-28) and ANF-(4-28), but little affinity to the truncated peptides ANF-(5-25) and ANF-(7-23). After treatment at pH 3.5 and 0 degrees C for 15 min, the membranes retained ANF-binding activity but with broader specificity, exhibiting high affinity to all four peptides above. It was suggested that an acidic metalloendopeptidase in the adrenal membranes may be involved in ANF receptor cleavage.
mesh_terms: D000302:Adrenal Cortex / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004492:Edetic Acid; D004533:Egtazic Acid; D006162:Guanylate Cyclase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008345:Manganese; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000187:drug effects*; D015032:Zinc
chemical_list: D011480:Protease Inhibitors; D011956:Receptors, Cell Surface; D008345:Manganese; D004533:Egtazic Acid; D009320:Atrial Natriuretic Factor; D004492:Edetic Acid; D008666:Metalloendopeptidases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D015032:Zinc