carboxypeptidase-b and preproenkephalin

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97-107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 microM CoCl2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.

Topics: Acetylcholinesterase; Animals; Carboxypeptidase B; Carboxypeptidases; Cattle; Eels; Enkephalins; Peptide Fragments; Protein Precursors; Trypsin

Trypsin and carboxypeptidase B-like (CPB-like) peptidases should be involved in processing proenkephalin to form the small biologically active enkephalins. A carboxypeptidase B-like activity from the soluble fraction of bovine adrenomedullary chromaffin granules which converts 125I-(Met)-enkephalin-Arg6 to 125I-(Met)enkephalin has previously been described and characterized (1,2). In this study, CPB-like activity in the membrane bound component of chromaffin granules is characterized and compared with that in the soluble fraction. Membrane and soluble CPB activities cleaved 125I-(Met)enkephalin-Arg6 or 125I-(Met)enkephalin-Lys6 to form 125I-(Met)enkephalin. Like the soluble enzyme, the CPB-like activity in the membrane component had a pH optimum of 6.0, was inhibited by thiol agents (PCMPSA, CuCl2) and metal ion chelators (EDTA, 1,10-phenanthroline), and was stimulated by Co++. The membrane CPB-like activity appeared to be an intrinsic membrane protein, since 80% of the activity remained with the membranes after washing with 1.0 M NaCl. Membrane and soluble CPB-like activities in chromaffin granules appear to be similar enzymes.

Topics: Adrenal Medulla; Animals; Carboxypeptidase B; Carboxypeptidases; Cattle; Chromaffin Granules; Chromaffin System; Enkephalins; Hydrogen-Ion Concentration; Intracellular Membranes; Protein Precursors