Activation mechanism of monkey and porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions

Abstract

The activation of Sepharose-bound monkey pepsinogen A under acidic conditions proceeded by cleavage of the L e~~~-I l e~' bond, indicating the occurrence of the intramolecular one-step activation, although the rate of cleavage was very slow. On the other hand the activation of monkey pepsinogen A in solution was highly dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation, indicating the predominance of intermolecular reaction. The cleavage site, however, was also restricted to the L e~~~-I l e~' bond. Thus, apparently exclusive one-step activation occurred in monkey pepsinogen. The activation of porcine pepsinogen A in solution was also dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation. The major cleavage site by the exogenously added pepsin was the L e~~~-I l e~~ bond. Therefore the site most susceptible to the intermolecular attacks was the bond connecting the activation segment and the pepsin moiety in both monkey and porcine pepsinogens. In porcine pepsinogen, however, a part of the zymogen was activated through the intermediate form, and an intramolecular reaction was suggested to be involved in the generation of this form.These results showed that in both pepsinogens A the intramolecular reaction occurred, first yielding pepsin A or the intermediate form, which then acted intermolecularly on the remaining pepsinogen or the intermediate form to complete the activation in a short time. A molecular mechanism for the activation reaction was proposed to explain consistently the experimental results.Pepsinogen is converted to pepsin under acidic conditions by releasing the so-called activation peptides from its NH2-terminal part. Two pathways are known for the activation, i.e. a one-step (direct conversion) pathway and a stepwise (sequential conversion) pathway. Monkey pepsinogen A has been shown to be activated nearly exclusively by the one-step pathway releasing the intact 47-residue activation segment [l] (see Fig. 1). This pathway has also been shown to occur in the activation of porcine [2], rabbit [3] and chicken [4] pepsinogens A, although in porcine and rabbit pepsinogens sequential pathways occurred simultaneously [2,3,5,6]. Contrary to these pepsinogens, the activation of bovine prochymosin [7], and human

Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation

“…(42) as a function of the initial pepsinogen concentration at different reaction times, using the values obtained for K 2 , k 3 and K 4 . The results obtained are in agreement with those of Kageyama and Takahashi (1987), who indicated that the intramolecular reaction is especially important in the ini-tial phase of the activation process to generate the active enzyme, and that the intermolecular reaction plays a significant role in acceleration and completion of the activation. However, as Z 0 is increased and at higher reaction times, r values are less than 1, indicating that the bimolecular reaction becomes more important under these conditions.…”

“…Physiological examples of these processes are the activation of prekallikrein to kallikrein (Magklara et al, 2003;Shariat-Madar et al, 2004), the conversion of trypsinogen to trypsin (Manjabacas et al, 1995;Liu and Wang, 2004) and the activation of pepsinogen to pepsin (Al-Janabi et al, 1972;Kageyama and Takahashi, 1987;Kageyama, 1988;Kageyama et al, 1989;Nielsen and Foltmann, 1993;Richter et al, 1998). Physiological examples of these processes are the activation of prekallikrein to kallikrein (Magklara et al, 2003;Shariat-Madar et al, 2004), the conversion of trypsinogen to trypsin (Manjabacas et al, 1995;Liu and Wang, 2004) and the activation of pepsinogen to pepsin (Al-Janabi et al, 1972;Kageyama and Takahashi, 1987;Kageyama, 1988;Kageyama et al, 1989;Nielsen and Foltmann, 1993;Richter et al, 1998).…”

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

“…Activation of immobilized pepsinogen is thought to be suitable to clarify intramolecular reactions. With Sepharose-bound porcine pepsinogen A, cleavage has been shown to generate pseudopepsin A [75,76], while pepsin A is generated with immobilized monkey pepsinogen A [83]. The involvement of intramolecular reactions might also be estimated in terms of excess substrate since intermolecular reactions between pepsinogens or between pepsinogen and pepsin may be hindered under this condition [84 -86].…”

“…Apart from two different activation pathways, two different cleavage reactions are possible for activation: in- Figure 4. The primary cleavage sites upon activation of various pepsinogens are summarized based on the results of pepsinogens A from human [80,87], monkey [12,73,83], pig [74,76], cattle [142], bear [69], house musk shrew [29], rabbit [13], and chicken [81]; cattle pepsinogen B [38]; progastricsins from human [77], monkey [12,83], pig [143], house musk shrew [29], and frog [22]; cattle prochymosin [78]; and tuna pepsinogens [144]. The residues in the activation segment are numbered according to the porcine pepsinogen A sequence and characterized by the suffix p. Residues in filled boxes are conserved in these sequences.…”

Development‐dependent expression of isozymogens of monkey pepsinogens and structural differences between them

“…(Tokyo, Japan) ; oligo(dT)-cellulose, the cDNAsynthesis kit, the pUC-cloning kit, and cloning vectors M23mp18 and M13mp19 (replicable forms) were obtained from Boehringer Mannheim GmbH (Mannheim, Federal Republic of Germany) cloning vector llgtlO and the DNA in vitro packaging kit (Gigapack Gold) were from Stratagene Cloning System (SCS) (La Jolla, CA, USA); the SequenaseTM DNA Table S2. Scmzniury of the activution of pepsinogen isoqmogens Activation was carried out at pH 2.0 and at pH 3.0. at 14"C, by the procedure described previously [40]. t s O (Pg) and tS0 (p) represent times needed for the disappearance of half of the initial amount of pepsinogen, and for the increase in the level of pepsin up to half of the equivalent amount of the initial pepsinogen, respectively.…”