Search

Human progastricsin was prepared from extracts of gastric mucosa by chromatography on columns of DEAE-cellulose. The amino acid compositions of progastricsin and gastricsin were determined and calculated on the basis of the molecular weights 38 000 and 32 000 respectively. The activation of progastricsin at pH 2 was investigated and monitored by agarose gel electrophoresis at pH 5.4. Two intermediates were observed. Determination of the amino acid sequence showed that the propart consists of 43 amino acid residues. A pronounced homology with other gastric zymogens was found. With the proenzyme amino acid residue numbering used previously [B. Foltman (1981) Essays in Biochemistry, 17, 52-84] the activation of progastricsin at pH 2 may be summarized as follows. The first cleavage occurs after Phe (p27). At pH 5.4 the peptide remains associated with the protein (intermediate I). Subsequent proteolysis removes the peptides from Leu (p28) to Leu (p45). At pH 5.4 the N-terminal peptide from progastricsin (p2-p27) remains associated with gastricsin (intermediate II) until the propart peptide is hydrolysed to smaller fragments.

Porcine pepsinogen A (EC 3.4.23.1) and progastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of progastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of progastricsin. The sequence of this was determined; in addition, the first 30 residues of gastricsin were sequenced. The sequence of the first 73 amino acid residues of progastricsin shows an overall identity with progastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pepsinogen A numbering) is changed to Arg in porcine progastricsin.

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2–3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S4), Leu 30 Val (S1/S3), His 74 Gln (S2′), Val 111 Ile (S1/S3), Lys 220 Met (S4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Development of the porcine gastric proteases (chymosin, pepsin A, B and C) has been studied in the fetal pig in the last third of gestation (term 115 days). The possibility that the prepartum rise in circulating cortisol is involved in gastric maturation was investigated by infusing immature fetuses with cortisol (osmotic minipumps implanted at 82-90 days of gestation). Concentrations of prochymosin in fundic tissue and stomach contents increased before term, correlated positively with logio plasma cortisol values (r = 0.68-0.76, p < 0.001), and were stimulated by cortisol infusion (p < 0.001). The pH of stomach contents decreased (from pH 7 to 3), correlated negatively with logio plasma cortisol values (r = -0.69, p < 0.001), and was reduced by cortisol infusion (p < 0.05). Only trace amounts of pepsinogens could be detected in fetal pigs. By immunohistochemistry, it was shown that cortisol increased the number and distribution of prochymosin-containing cells in the fundic gland. Stimulating effects were also observed for the small populations of pepsinogen-reactive cells present in some of the fetal pigs. The results suggest that endogenous cortisol stimulates the rise in prochymosin synthesis and secretion together with increased gastric acidity in the prenatal period of the pig.

Reaction products formed during activation of porcine pepsinogen A at pH 2 were characterized by native agar-gel electrophoresis and by denaturing SDS/PAGE. The results revealed the presence of non-covalent intermediates between prosegment peptides and pepsin. The complexes Leu1p-Leu44p/pepsin and Leu1p-Leu16p/pepsin were isolated (the prosegment residues are characterized by the suffix p; numbering of residues starts again from the N-terminus of pepsin). Relative to mature pepsin, the inherent milk-clotting activities of the intermediates were 3% and 18%, respectively. The intermediates were incubated at pH 8.5 for 20 min at 28 degrees C and the residual proteolytic activities were tested at pH 2. The stabilities at pH 8.5 were between those of pepsinogen and pepsin, Leu1p-Leu44p/pepsin being most stable. The implications of these findings for the conformational changes that occur during the activation of pepsinogen are discussed.

Cheesemaking and fermentation represent the first examples of applied biochemistry and biology. Whereas living microorganisms are used in fermentation processes, the clotting of milk for cheesemaking has always required soluble enzymes. The milk-clotting enzyme from the fourth stomach of the calf was one of the first enzymes of which purification was attempted, and Deschamps1 suggested the name chymosin, derived from the Greek word for gastric liquid ’chyme’. This designation was later used in continental European languages, whereas in English the name rennin, derived from rennet, was used.2 Misunderstandings often occurred between rennin and renin from the kidneys, and therefore the designation chymosin was recently adopted in English3 and it is now used in the recommended international enzyme nomenclature.4

The relationship between male infertility and the pepsinogen C content in semen has been investigated. The activation of the seminal pepsinogen C in the vagina has been studied under physiological conditions. Samples of semen from 48 vasectomized males and from 46 males of infertile couples were analyzed for pepsinogen C by radioimmunoassay. No correlation was found between the level of pepsinogen C and seminal characteristics, including sperm concentration, motility, and morphologic features. The mean concentration of pepsinogen C was 42.2 micrograms/ml; the first, second, and third quartile were 18.4, 29.6, and 57.6 micrograms/ml, respectively. No significant difference in the level of pepsinogen C was observed between semen of normal quality, semen of reduced quality, and semen with aspermia. Activation of pepsinogen C occurred within 3 h when semen was incubated at pH below 5.0 at 37 degrees C. Intravaginal activation was investigated in six experiments in which semen from two males was instilled in three females. In four experiments with two couples, post-coital activation was investigated. Pepsin C activity in vaginal fluid was detected an average of 3 h (range 2-5 h) and 5 h (4-7 h) after instillation or ejaculation, respectively. Vaginal pH had then been below 4.5 for approximately 1 h. Pepsin C activity was present in the vagina for more than 24 h thereafter. It is most likely that seminal pepsin C is without influence on the fertilizing spermatozoon. However, pepsin C may exert a local effect in the vagina by degrading seminal proteins, thus preventing an immunogenic response in females.

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and car chymosin was obtained after acid activafion of the zymogen. The enzyme showed the optimum of ganeral proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.

1. 1. Calculated as active enzymes the gastric mucosa of piglets contains 1–4 mg of chymosin (EC 3.4.23.4) per g of mucosa during the first week of life. No chymosin is present in pigs older than 2 months. 2. 2. The production of pepsin (EC 3.4.23.1) is very low during the first week of life; a rapid increase occurs after about 3 weeks. 3. 3. Pig chymosin and pig pepsin have higher milk clotting activity against porcine milk than against bovine milk. 4. 4. Neonatal proteinases with high milk clotting and low general proteolytic activities are probably important for postnatal uptake of immunoglobulins.

The complete amino acid sequence of calf chymosin (rennin) (EC 3.4.23.4) has been determined. The sequence consists of a single peptide chain of 323 amino acid residues. The primary structure of the precursor part of calf prochymosin was published previously (Pedersen, V.B., and Foltmann, B. (1975) Eur. J. Biochem. 55, 95-103), thus we are now able to account for the total 365 amino acid residues of calf prochymosin. Comparison of the sequence of calf prochymosin with that of pig pepsinogen A (EC 3.4.23.1) shows extensive homology. In the precursor part of the sequence, 15 residues are located at identical positions, as compared to 189 identical residues in the respective enzymes. Furthermore comparison to Penicillium janthinellum acid proteinase (penicillopepsin) (EC 3.4.23.7) shows that 76 residues are common to this enzyme and to the two gastric proteinases. These homologies in sequence further suggest that the folding of the peptide chain in chymosin is very similar to that of other acid proteinases.

A rapid purification procedure of thiol:protein-disulfide oxidoreductase (EC 1.8.4.2) from bovine liver has been developed. The procedure is based on that of D. F. Carmichael, J. E. Morin, and J. E. Dixon (1977, J. Biol. Chem. 252 , 7163–7167), and contains the following steps: homogenization in Triton X-100, selective heat denaturation, chromatography on CM-Sephadex C-50, and chromatography on DEAE-Sephadex A-50. The final preparation has a high specific activity and a high level of purity as judged by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

Bovine pepsinogen is converted into pepsin by removal of 45 amino acid residues from the NH2 terminus of the single polypeptide chain. The complete sequence of the activation peptides has been deduced from two overlapping sets of peptides, one set obtained by autoactivation of the zymogen at pH 2 and the second set from a tryptic digest of maleylated pepsinogen. A peptide derived from the 17 residues at the NH2 terminus of bovine pepsinogen has been identified as an inhibitor of the milk-clotting action of pepsin. Extensive sequence homologies exist among the activation peptides of bovine and porcine pepsinogens and bovine prochymosin. In the peptides from the two pepsinogens, 30 of the 44 or 45 residues are identical, and most of the substitutions in the other residues are conservative. The preservation of positive charges in the NH2-terminal portion of the zymogen molecules, lost during activation, agrees with current ideas on the involvement of this segment in stabilizing the physical structure of the zymogens.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.

Antiserum raised against an erythrocyte membrane-attached aspartic proteinase precipitates a non-pepsin gastric proteinase. With a monospecific antiserum raised against the non-pepsin gastric proteinase the two enzymes show immunochemical identity. The isoelectric points of both are between 4.5 and 4.6. By SDS-polyacrylamide gel electrophoresis the two proteinases behave the same way. Under non-reducing conditions the main components show molecular weights around 90 000 and after reduction about 58 000. The proteinase may tentatively be classified as cathepsin E.

Chymosin (EC 3.4.4.3) is the predominant protease in the fourth stomach of the young calf. For many years the term rennin has been used in English literature, but since mistakes often occur between rennin from the calf stomach and renin (EC 3.4.4.15) from the kidneys, we return to the older term chymosin, which previously has been used in European continental literature. Like other proteases from the digestive tract chymosin is secreted as an inactive precursor, which is transformed into active enzyme by a limited proteolysis. The chemistry of the enzyme and the kinetics of the activation process have been reviewed a few years ago [ 1 ] . This communication describes isolation and analyses of some peptides obtained after tryptic digestion of prochymosin in which the amino groups were blocked by reaction with maleic anhydride, and it is shown that these peptides constitute the N-terminal and the C-terminal amino acid sequences of the zymogen.

By conversion of prochymosin into active chymosin an N-terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology.

Antisera were raised in rabbits against chromatographically purified preparations of pepsin and gastricsin. With these antisera the contents of pepsin and gastricsin in gastric juice were determined by rocket immunoelectrophoresis. The potential content of pepsin and gastricsin of a secondary standard of gastric mucosal extract was calibrated against the chromatographically purified enzymes. This secondary standard was used for routine analyses. The intra-assay and between-assay precision was 3–4% and 6–9%, respectively. Ten healthy volunteers underwent a standard pentagastrin test. The amounts of pepsin and gastricsin determined by rocket immunoelectrophoresis corresponded to the amounts observed by ion exchange chromatography of gastric juice. After stimulation with pentagastrin the secretion of both pepsin and gastricsin was increased about 10 times.

The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor.

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The decuced amino sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acide residues with a total molecular weight of 38,701. Clusters of idendtities around the active site cleft support the assumption that these proteinasess have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.

Two papain inhibitors, I1 and I2, from rat skin extract were purified by affinity chromatography on KSCN-modified papain-agarose gel and by gel filtration on Sephadex G-100. I1 had a molecular weight of 74 000, a pI of 4.6, and it contained 4% of carbohydrates. I1 inhibited papain, ficin, bromelain, rat skin benzoylarginine-2-naphthylamide hydrolase, and to a minor extent, rat skin cathepsin C and bovine trypsin. Bovine chymotrypsin or rat skin cathepsin D were not inhibited and benzoylarginine-2-naphthylamide hydrolase was inhibited only at alkaline pH. An inhibitor corresponding to I1 was present in various rat tissues and also in serum. A similar inhibitor was present in the skin of cat, rabbit, guinea pig, and man. I2 had a molecular weight of 13 400, a pI of 4.9 and it contained no carbohydrates. I2 inhibited all thiol proteases tested, but not trypsin, chymotrypsin, or rat skin cathepsin D. I2 formed an equimolar complex with papain and benzoylarginine-2-naphthylamide hydrolase. I2 was present in rat skin, muscle, lung, and small intestine, but not in kidney, liver, or serum. A similar inhibitor was found in skin extracts of cat, rabbit, guinea pig, and man.

The N-terminal amino acid sequences of dogfish pepsinogen (8 residues), seal pepsinogen (26 residues) and bovine pepsinogen B (25 residues) have been determined. The primary structures of all three pepsinogens show considerable homology with those of zymogens of gastric proteases sequenced previously.

1. 1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 3. 3. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. 4. Lamb prochymosin and pepsinogen are both glycosylated.

The proteinases of the gastric juice are secreted as inactive precursors which are irreversibly converted into the usually known enzymes by limited proteolysis releasing from 42-45 amino acid residues [l-4]. To initiate this process no other activating agents than hydrogen ions are required. It is generally accepted today that the initial stage in the activation process is a conformational change of the zymogen molecule from inactive to active conformation [5-81, but for the subsequent pathway of the activation, different views have been reported. Bustin and Conway-Jacobs [9] coupled porcine pepsinogen A to Sepharose in such concentrations that interactions between individual pepsinogen molecules would be very limited. After exposure to acid, active enzyme and new N-terminal isoleucine were observed. Al-Janabi et al. [lo] showed by activation of porcine pepsinogen A in solution at pH 2 that the conversion of alkali-stable pepsinogen to an alkali-labile product follows a first order reaction. Sanny et al. [ 111 found that the initial cleavage product formed during activation at pH 2 had the N-terminal sequence Ile-Gly-. They concluded that during activation of porcine pepsinogen at pH 2 the peptide’bond Leu44-Ile4s is the first to be cleaved. This is in contrast to experiments of Dykes and Kay [12]. At pH 2.5 they activated porcine pepsinogen in presentof the inhibitor pepstatin [ 131 and from the ,mino acid composition of the peptides produced during the reaction they inferred that peptide bond Leur6-he,, was cleaved first. For the intermediate (which was inhibited in their experiments) they suggested the name ‘pseudopepsin’. The experiments reported in this paper confirm that at pH 2 the predominant initial cleavage occurs at peptide bond 16-l 7. This cleavage is the results of an intramolecular reaction and we show that the pseudopepsin has proteolytic activity and is alkali-labile.

Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105.

Preparations of pig kidney histaminase were analyzed for total compositions, N-terminal residues, patterns of tryptic peptides and for subunits under dissociating conditions. The crystalline enzyme is pure by several criteria but not completely homogeneous. It is rich in acidic or amidated residues and has a low content of cysteine/halfcystine. Preparation analyzed consistently contained a few percent of carbohydrate. Subunits are concluded to be identical or highly similar with a molecular weight of about 90 000, which is consistent with other analyses.

Proteinases with a pH-optimum of general proteolytic activity at pH below 6 are called acidic proteinases. Such enzymes were at first recognized as the proteinases of the vertebrate gastric juice, and for many years porcine pepsin and calf chymosin have been the most well-known members of this group.