Abstract
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.
Highlights
Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene
On the basis of earlier studies we developed a rapid method for isolation of cationic trypsin from pancreatic juice
Our comparative studies on native human cationic trypsin and the unmodified native and recombinant forms show that the modification does alter neither the amidolytic nor the esterolytic activity of the enzyme (Table I)
Summary
Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. We determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. To model the mutation of human cationic TG at position 21, Thr of rat anionic TG was replaced with Asn or Ile. It was found that Ile at position 21 significantly decreased the rate of autoactivation and zymogen degradation without affecting trypsin activity and stability [8]. It has been shown that the Asn to Ile mutation in the human cationic TG, contrary to the effects observed in rat anionic TG, did not change the stability of the zymogen [9]. We isolated native human cationic trypsin and hPSTI from human pancreatic juice, compared the enzymic properties of native and recombinant tis-associated N21I mutant of human cationic trypsinogen; hPSTI, human pancreatic secretory trypsin inhibitor; Z-GPR-pNA, N-carbobenzyloxy-glcyl-prolyl-arginyl p-nitroanilide; Z-K-SBzl, N␣-carbobenzyloxy-lysine thiobenzylester; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis
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