IMMUNOLOGICAL STUDIES ON PEPSIN AND PEPSINOGEN

1. Alkali (pH 7.6)-denatured pepsins from swine, cattle, and guinea pigs precipitate in swine pepsin antiserum. Similarly treated pepsins from the rabbit, chicken, and shark do not. 2. Pepsin antisera react with both pepsin and pepsinogen, but do not react with the serum proteins from the homologous species. 3. Pepsinogen antisera react with pepsinogen, but not with twice crystallized pepsin, nor with the serum proteins from the homologous species. Positive reactions between activated pepsinogen and pepsinogen antiserum have been observed. It was possible to remove the reacting material from either the pepsinogen or the activated pepsinogen mixture. 4. Antisera made with serum proteins do not react with the homologous pepsin or pepsinogen.     

Evidence for the occurrence of one-step activation in porcine pepsinogen

Upon activation at pH 2.0 and 14°C, a significant portion of porcine pepsinogen was found to be converted directly to pepsin, releasing the 44-residue intact activation segment. The released segment was further cleaved to smaller peptides at pH 2.0, but at pH 5.5 it formed a tight complex with pepsin, and the complex was chromatographically indistinguishable from pepsinogen. This intact segment could be isolated for the first time. Thus one-step activation occurs in porcine pepsinogen along with the already known sequential activation.     

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Primary structure,unique enzymatic properties,and molecular evolution of pepsinogen B and pepsin B

Purification of pepsinogen B from dog stomach was achieved. Activation of pepsinogen B to pepsin B is likely to proceed through a one-step pathway although the rate is very slow. Pepsin B hydrolyzes various peptides including beta-endorphin, insulin B chain, dynorphin A, and neurokinin A, with high specificity for the cleavage of the Phe-X bonds. The stability of pepsin B in alkaline pH is noteworthy, presumably due to its less acidic character. The complete primary structure of pepsinogen B was clarified for the first time through the molecular cloning of the respective cDNA. Molecular evolutional analyses show that pepsinogen B is not included in other known pepsinogen groups and constitutes an independent cluster in the consensus tree. Pepsinogen B might be a sister group of pepsinogen C and the divergence of these two zymogens seems to be the latest event of pepsinogen evolution.     

Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0. Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37 degrees C and the activity under these conditions was comparable to that of pepsin.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Cloning of the authentic bovine gene encoding pepsinogen a and its expression in microbial cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.

A new pepsinogen component, pepsinogen C, was purified from the gastric mucosa of Japanese monkey. The chromatographic behavior of this component on DE-32 cellulose was coincident with that of pepsinogen III-2 previously reported (1), and final purification was performed by large-scale polyacrylamide disc gel electrophoresis. The molecular weight was 35,000 as determined by gel filtration. The ratios of glutamic acid to aspartic acid and of leucine to isoleucine were higher than those of other Japanese monkey pepsinogens. The activated form, pepsin C, had a molecular weight of 27,000 and contained a large number of glutamic acid residues. The optimal pH for hemoglobin digestion was 3.0. Pepsin C could scarcely hydrolyze the synthetic substrate, N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT). 1, 2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester (DAN) inhibited pepsin C [EC 3.4.23.3] in the same way as pepsin III-3 of Japanese monkey. The susceptibility to pepstatin of pepsin C was lower than that of pepsin III-3, and 500 times more pepstatin was required for the same inhibitory effect. The classification and nomenclature of Japanese monkey pepsinogens and pepsins are discussed.     

Isolation of an activation intermediate and determination of the amino acid sequence of the activation segment of human pepsinogen A

Upon activation of human pepsinogen A at pH 2.0 in the presence of pepstatin, an intermediate form was generated together with pepsin A. This activation intermediate could be separated from pepsinogen A and pepsin A by DE-32 cellulose chromatography at pH 5.5. It had a molecular weight intermediate between those of pepsinogen A and pepsin A, and contained about half the number of basic amino acid residues in pepsinogen A. It had phenylalanine as the amino(N)-terminal amino acid, and was deduced to be generated by release of N-terminal 25 residue segment from pepsinogen A. Amino acid sequence determination of the N-terminal portions of pepsinogen A and the intermediate from enabled us to elucidate the entire acid sequence of the 47-residue activation peptide segment as follow: [Formula: see text]. On the other hand, upon activation of pepsinogen A at pH 2.0 in the absence of pepstatin, cleavage of the activation segment occurred at several additional bonds. In addition, upon activation both in the presence and in the absence of pepsitatin, an additional activation intermediate, designated pepsin A', was formed in minor quantities. This form was identical with pepsin A, except that it had an additional Pro-Thr-Leu sequence preceding the N-terminal valine of pepsin A.     

Regulation of interleukin-1 production in murine macrophages and human monocytes by a normal physiological constituent

The in vitro production of large quantities of interleukin-1 (IL-1) in mouse peritoneal exudate macrophages and human peripheral blood monocytes is possible through the use of the proteolytic enzyme pepsin and its zymogen pepsinogen. Equal amounts of IL-1 are generated by pepsin in the absence or presence of polymixin B. The addition of pepsin or pepsinogen had no effect on the proliferation of C3H/HeJ thymocytes to the plant mitogen phytohemagglutinin. Pepsin and pepsinogen are present in significant quantities in immune cells and the plasma. Although little is known concerning the physiological role of pepsin and pepsinogen outside of the gastrointestinal system, it may be proposed that the in vivo production of IL-1 may in part be regulated by the cellular and plasma concentrations of pepsin and pepsinogen.     

Semen-induced ovulation in the bactrian camel (Camelus bactrianus)

Bactrian camels (63 female female, 8 male male) were used in the breeding season to determine the factors that will induce ovulation. After insemination of semen samples into the vagina, the ovaries were checked for ovulation by rectal palpation. The results indicated that ovulation was induced by the seminal plasma, but not by the spermatozoa, and the incidence of ovulation after insemination was 87%. Most of the females (66%) had ovulated by 36 h after insemination and the rest by 48 h, as after natural service. The least amount of semen required to elicit ovulation was about 1.0 ml. Intramuscular injections of LH, hCG and LHRH also caused ovulation, even in females that had not ovulated in response to insemination.     

Pepsin D: A minor component of commercial pepsin preparations

Methods are described for the isolation and purification of pepsin D, an enzyme which accounts for about 10% of the enzymic activity in commercial preparations of pepsin. Pepsin D is similar to pepsin in having a molecular weight of about 35000, the same C-terminal amino acid sequence, and an N-terminal isoleucine residue. It differs in having no phosphate residue. Pepsin D is similar to pepsin in its ability to digest haemoglobin, acetyl-l-phenylalanyl-l-di-iodotyrosine and gelatin but it is twice as active as pepsin in the clotting of milk. It has the same specificity as pepsin in its action on the B-chain of oxidized insulin. It is probable that the pepsin D in commercial preparations of pepsin arises from the activation of gastric pepsinogen D.     

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin

When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.     

Studies on the irreversible step of pepsinogen activation

The bond cleavage step of pepsinogen activation has been investigated in a kinetic study in which the denatured products of short-term acidifications were separated on SDS-polyacrylamide gels and the peptide products were quantitated by densitometry. Although several peptide products were observed, under the conditions of the experiments (pH values between 2.0 and 2.8, 22 degrees C), the only one that was a product of an initial bond cleavage was the 44-residue peptide, which upon removal from pepsinogen yields pepsin. The rate constant for this bond cleavage is 0.015 s-1 at pH 2.4, which is the same as that at which the alkali-stable potential activity of pepsinogen had been found to convert to the alkali-labile activity of pepsin. When the conversion of zymogen to enzyme was followed by the change in fluorescence of adsorbed 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS), the rate of change in TNS fluorescence was the same as the conversion to alkali lability. However, pepstatin blocked the bond cleavage of pepsinogen to pepsin, but it permitted the fluorescence change to proceed. In fact, it accelerated the apparent rate of change of TNS fluorescence by shifting the pKa of an essential conjugate acid from 1.7 to 2.6. The conversion to alkali lability, therefore, may be considered to be a composite of a relatively slow conformational change (at the measured rate), followed immediately by a relatively fast bond cleavage.     

Occurrence of two different pathways in the activation of porcine pepsinogen to pepsin

Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the released 44-residue segment was direct evidence of this one-step process. At pH 5.5 the segment bound tightly to pepsin to form a 1:1 pepsin-activation segment complex, which was chromatographically indistinguishable from pepsinogen. The other is a stepwise-activating or sequential pathway, in which pepsinogen is activated to pepsin through intermediate forms, releasing activation peptides stepwisely. These intermediate forms were isolated and characterized. The major intermediate form was shown to be generated by removal of the amino-terminal 16 residues from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17-44, and hence the stepwise-activating process was deduced to be mainly a two-step process.     

Conformational instability of the N- and C-terminal lobes of porcine pepsin in neutral and alkaline solutions.

Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two-chain pepsin or a two-chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257-17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline-inactivated pepsin was regenerated by the addition of the recombinant N-terminal lobe but not by the C-terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N-terminal lobe. A complex between recombinant N-terminal lobe of pepsinogen and alkaline-denatured pepsin has been isolated. This complex is structurally similar to a two-chain pepsinogen, but it contains an extension of a denatured pepsin N-terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline instability of the N-terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N-terminal domain (as compared to 19 in the C-terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118.(ABSTRACT TRUNCATED AT 250 WORDS)