title: The preparation and properties of the catalytic subunit of bovine enterokinase.
abstract: A limited reduction of the disulfide bonds of bovine enterokinase (enteropeptidase, EC 3.4.21.9) was accomplished with 50 mM dithioerythritol, at pH 9.0, and at 4 degrees C. The conditions separated the heavy and light subunits quantitatively with improved reliability when compared to the conditions used previously (Savithri, H. S., and Light, A. (1980) Biochim. Biophys. Res. Commun, 94, 360-365). Pancreatic trypsin inhibitor was added to the reaction to ensure that the yield of the heavy subunit was equal to that of the catalytic subunit (light subunit). Otherwise the heavy subunit was subject to extensive degradation. The subunits were alkylated with iodoacetate and then resolved on Sephadex G-150. Amino acid analyses and the incorporation of [14C]carboxymethyl groups showed that 3.1 carboxymethylcysteine residues were in the catalytic subunit and 8.9 in the heavy subunit. The catalytic subunit had normal catalytic activity toward N-benzoyl-L-arginine ethyl ester, enhanced activity toward N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester, and lower activity toward N-benzoyl-DL-arginine p-nitroanilide. The catalytic subunit retained the restricted specificity of intact enterokinase, but the rate of activation of trypsinogen was much slower. It is likely that the limited reduction of the disulfide bonds of the catalytic subunit altered the interaction of protein substrates with the specificity site.
mesh_terms: D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000032:analysis; D004386:Duodenum / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification*; D004765:Enteropeptidase / Q000302:isolation & purification* / Q000378:metabolism; D007413:Intestinal Mucosa / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; D010450:Endopeptidases; D004765:Enteropeptidase