Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.     

Characterization of fish acid proteases by substrate–gel electrophoresis

Several analytical techniques based upon the use of substrate–polyacrylamide gel electrophoresis were evaluated to achieve characterization of aspartate proteases in fish stomach. Since aspartate proteases of fish are more stable at high pH than mammalian pepsins, the most accurate technique for activity assessment is electrophoresis at neutral pH and revealing of such activity at low pH with hemoglobin as substrate. The technique is suitable for characterization of proteases and in comparative assessment of acid protease activity in different sparids.     

Purification and characterization of pepsinogens from the gastric mucosa of African coelacanth, Latimeria chalumnae, and properties of the major pepsins

Two major pepsinogens, PG1 and PG2, and one minor pepsinogen, PG3, were purified from the gastric mucosa of African coelacanth, Latimeria chalumnae (Actinistia). PG1 and PG2 were much less acidic than PG3. Their molecular masses were estimated by SDS-PAGE to be 37.0, 37.0 and 39.3 kD, respectively. When incubated at pH 2.0, PG1 and PG2 were converted autocatalytically to the mature pepsins through an intermediate form, whereas PG3 was converted to an intermediate form, but not to the mature pepsin autocatalytically. The N-terminal sequencing indicated that the 42 residue sequences of the propeptides of PG1 and PG2 were essentially identical with each other, but different from that of PG3. A phylogenetic tree based on the N-terminal propeptide sequences indicates that PG1 and PG2 belong to the pepsinogen A group, and PG3 to the pepsinogen C group. From the phylogenetic comparison, coelacanth PG1 and PG2 appear to be evolutionally closer to tetrapod pepsinogens A than ray-finned fish pepsinogens A, consistent with the traditional systematics. Pepsins 1 and 2 were essentially identical with each other and rather similar to mammalian pepsins A in the pH optimum toward hemoglobin (pH 2-2.5), the cleavage specificity toward oxidized insulin B chain and strong inhibition by pepstatin, except that they possessed a significant level of activity in the higher pH range unlike mammalian pepsins A.     

A pepsinogen from rainbow trout

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.     

The pathway of pepsin-catalysed transpeptidation. Evidence for the reactive species being the anion of the acceptor molecule

Several minor pepsinogens, present in extracts of bovine fundic mucosa obtained from the fourth stomach or abomasum, were separated from the main pepsinogen by chromatography on hydroxyapatite at pH7.3. The major pepsinogen and two of these minor pepsinogens were studied in detail. All three zymogens have N-terminal Ser-Val-, C-terminal -Val-Ala and not more than 1mol of glucose/mol of protein; no significant differences in amino acid composition were found. The pepsinogens differ in their organic phosphate content, which accounts for their chromatographic separation. By activation at 0 degrees C and pH2, a corresponding series of pepsins is formed. These enzymes were separated by hydroxyapatite chromatography at pH5.7. All the pepsins have N-terminal valine, C-terminal alanine and are free from carbohydrate. Again the only difference detected among them is in their organic phosphate content. The pepsins of high phosphate content are converted by an acid phosphatase in vitro into pepsins of low phosphate content.     

Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.     

Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis)

Two pepsins (A and B) were purified from the stomach of pectoral rattail (Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 °C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0–6.0 but were unstable at the temperatures greater than 40 °C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0–30%). The enzymes had high affinity and activity toward hemoglobin with K_m and K_cat values of 98–152 μM and 32–50 S^− 1, respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.     

Comparison of pig and chicken pepsins for protein hydrolysis.

The aim of the present study was to compare the degree of proteolysis with pig (PP) and chicken (CP) pepsins in order to find out whether PP can be used instead of CP to simulate gastric hydrolysis in the chicken. First, the pH activity profile of the two pepsins was compared using three substrates. For haemoglobin, CP showed a slightly higher optimal pH than PP, 2.5-3 and 2, respectively. For two plant protein sources (peas, wheat), the optimal pH was similar for the two enzymes, about pH 1.5. For the three substrates tested, CP exhibited a high level of activity over a broader pH range than PP. Second, the susceptibility of the two plant proteins to hydrolysis by each of the two pepsins was studied at pH levels near the chicken gastric pH (1.5-3.5). For PP, pea proteins were hydrolysed more than wheat ones, while, for CP, the hydrolysis was dependent on pH. Therefore, the classification of the two studied protein sources was dependent on the enzyme species and pH. The results of this study show that the choice of in vitro hydrolysis conditions to assess the digestibility of proteins must be made with great care.     

Demonstration of chymosin (EC 3.4.23.4) in the stomach of newborn pig.

The stomach of newborn pig contains a proteinase that is immunologically closely related to calf chymosin (rennin) (EC 3.4.23.4.). None of the pepsins from the stomach of adult pig is present in the newborn pig. Pig chymosin has optimal general proteolytic activity around pH 3.5. The ratio of milk-clotting activity to general proteolytic activity is about 30--70 times higher than that of pyloric and fundic pepsins.     

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.     

On the activation of the canine pepsinogens.

Acidification induces a conversion of canine pepsinogens by a sequential mechanism to the active pepsins. Activation in the presence of pepstatin, which strongly inhibits the pepsins but does not prevent the first step of activation, allows the isolation of the peptide released in this first step. This peptide inhibits the milk clotting activity of canine and also porcine pepsin. Canine pepsins obtained in the absence of pepstatin were characterized by amino acid composition, molecular weight, and activity against hemoglobin and milk and compared with those of other mammalian pepsins.     

Purification and characterization of goat pepsinogens and pepsins

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH₂-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose.

A procedure was devised for isolating human pepsins 1, 2, 3 and 5 from gastric juice by repetitive column chromatography on DEAE-cellulose. The combined yields in four different experiments varied from 14% to 90% of the total peptic activity of the starting material. The isolated individual pepsins were shown to behave as single homogeneous proteins on agar-gel electrophoresis at pH 5.0 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. There is preliminary evidence, requiring further study, of two other pepsins, one migrating between pepsins 1 and 2 and the other a pepsin-3 component associating closely with pepsin 5 on chromatography.     

Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes.

Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes.     

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family

We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.     

Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.     

Ovalbumin digestion by human pepsins 1, 3 and 5.

1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4--1.5 for pepsin 3 and pH 1.2--1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.     

The pepsins from human gastric mucosal extracts

1. The pepsins and pepsinogens of the gastric mucosal extracts of two normal subjects, of seven patients with gastric adenocarcinoma and of two patients with duodenal ulcer have been investigated by agar-gel electrophoresis and by ion-exchange chromatography. 2. Of the eight zones of proteolytic activity that have previously been reported in normal human gastric juice, seven can be detected in activated fundic mucosal extracts. Of these seven, four can be attributed to discrete pepsins, numbered 1, 3a, 3 and 5. 3. Zone 7 results from the activity of one or more enzymes that are alkali-stable and are best referred to as gastric proteinases rather than as pepsins. Zone 7 is much more evident in mucosal extracts than in gastric juice. 4. Zones 4 and 6 may result respectively from the activity of a pepsin-inhibitor complex and of an unactivated zymogen. 5. It was not possible, by the chromatographic methods employed, to separate satisfactorily the individual pepsins from activated extracts or their precursors from unactivated extracts, so that the ascribing of a pepsin to a specific zymogen must be considered tentative. Even so, pepsin 3 appears to arise from at least two major precursors, if not from three, whereas pepsins 1 and 5 each arise from a single major precursor. 6. Pyloric mucosal extracts contain principally zone 5 but also zones 6 and 7. These zones in general behave similarly to the corresponding zones of fundic extracts, but pyloric pepsin 5 migrates slightly faster on agar-gel electrophoresis than does fundic pepsin 5 and is a different enzyme. Zones 1 to 4 are absent.