SUBCELLULAR-LOCALIZATION, SUBSTRATE-SPECIFICITY AND CRYSTALLIZATION OF DUODENASE, A POTENTIAL ACTIVATOR OF ENTEROPEPTIDASE

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial c ells and ducts of Brunner's glands by the A-gold immunocytochemical me thod. Duodenase exhibits trypsin-like and chymotrypsin-like specificit ies with a preference fur substrates having lysine at the P1 and proli ne at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k(cat)/K- m of 35000 M-1 s(-1)) and alpha-N-serylprolyllysine 4-nitroanilide (K- cat/K-m of 2600 M-1 s(-1)), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its cata lytic properties and sites of localization, duodenase has been postula ted to bet an activator of the enteropeptidase precursor. A tetradecap eptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, a nd kinetic parameters of its hydrolysis by duodenase were determined ( K-m of 87 mu M; k(cat) of 1.4 s(-1); k(cat)/K-m of 16000 M-1 s(-1)). C rystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.