Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.
Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.
Szilágyi
L
L
Department of Biochemistry, Eötvös Loránd University, Puskin u 3, Budapest, H-1088, Hungary. szilagyi@cerberus.elte.hu
Kénesi
E
E
Katona
G
G
Kaslik
G
G
Juhász
G
G
Gráf
L
L
D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D002384:Catalysis; D002401:Cathepsin B / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000378:metabolism; D010195:Pancreatitis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000494:pharmacology; D014362:Trypsinogen / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D007532:Isoleucine; D014359:Trypsin Inhibitor, Kazal Pancreatic; D001216:Asparagine; D014362:Trypsinogen; D014357:Trypsin; D002401:Cathepsin B