Purification and characterization of a putative proenkephalin cleaving enzyme.
A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 mM), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different Km and Vmax values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower Km and Vmax of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.
Paik
S R
SR
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Lewis
R V
RV
D000311:Adrenal Glands / Q000201:enzymology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D010446:Peptide Fragments; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases; C060973:membrane-bound adrenal trypsin-like enzyme; D014357:Trypsin