Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

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.     

Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.     

Amino acid sequence of endothiapepsin. Complete primary structure of the aspartic protease from Endothia parasitica

The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined. The enzyme consists of 330 residues. The sequence determination was performed exclusively at the protein level. The homology of this fungal milk-clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25-30% identity. The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.     

Novel chromophore substrates of aspartyl proteinases

Chromophore substrates Dnp-Ala-Glu-Phe-Ala-Arg-NH2 and Dnp-Ala-Ala-Phe-Nle-Ala-Arg-NH2 of aspartic proteases were synthesized by a combination of chemical and enzymic methods. The kinetic parameters of their hydrolysis with pepsin, aspergyllopepsin, and chymosin were determined. The introduction of Nle in the P1' position gives stable enzyme-substrate complexes with pepsin and chymosin. A Glu residue at the P2 position contributes significantly to an increase in kcat for the chymosin hydrolysis.     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.     

X-ray study of chymosin. I. Molecular replacement at a 3 angstroms resolution

The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected.     

Various aspects of structural studies of aspartate proteinases

The paper is a brief account of aspartic proteinases' structural studies developed in V.A. Engelhardt Institute of Molecular Biology during the last 3 years. The work on porcine pepsin has been finalized after the refinement of the monoclinic crystal form at 1.8 A resolution performed in collaboration with the group of protein structure and function studies of the University of Alberta in Canada. An important structural property of chymosin which explains the enzyme specificity has been found. Protein engineering work on chymosin is being developed. The structural template for aspartic proteinases has been elucidated and on the basis of this template the model of HIV-1 protease molecule has been built. Some approaches to the design of HIV-1 protease inhibitors were elucidated.     

Isolation and characterization of porcine beta-casein.

Porcine beta-casein was isolated by chromatography on DEAE-cellulose. The protein had a molecular weight of 24 900 as determined by gel filtration on Sephadex G-100 in guanidine-HCl. Its amino acid composition differed from bovine beta-casein especia-ly in respect to serine, alanine and leucine. In common with bovine beta-casein the N-terminal amino acid was arginine; the C-terminal was either alanine or valine, while the C-terminal of bovine beta-casein is valine. At any temperature porcine beta-casein was more sensitive to Ca2+ than bovine beta-casein, while at a fixed Ca2+ concentration porcine beta-casein aggregated at a lower temperature than bovine beta-casein. Porcine beta-casein was susceptible to hydrolysis by calf chymosin but the proteolytic specificity differed from that of calf chymosin on bovine beta-casein.     

Evolution in the structure and function of aspartic proteases

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.     

X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.     

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.     

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Amino acid sequences around 1, 2-epoxy-3-(p-nitrophenoxy)propane-reactive residues in rhizopus chinensis acid protease: homology with pepsin and rennin.

Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site.     

A Review of: “Laboratory Techniques in Biochemistry and Molecular Biology Vol. 1, T. S. Work and E. Work (eds.) North Holland Publishing Co., Amsterdam/ American Elsevier Publishing Co. Inc., New York, 1969; 573 + vi pages,indices, hard cover; $29.00”

When calf rennet containing ～ 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorhed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain > 97% pure chymosin. The fraction that passed through unadsorbed was found to contain > 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzvme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Accessing the reproducibility and specificity of pepsin and other aspartic proteases

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from > 30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5–6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Characterisation of the barrier caused by luminally secreted gastro-intestinal proteolytic enzymes for two novel cystine-knot microproteins

It was the aim of this study to evaluate the stability of two novel cystine-knot microproteins (CKM) SE-ET-TP-020 and SE-MC-TR-020 with potential clinical relevance towards luminally secreted proteases of the gastrointestinal tract in order to gain information about their potential for oral administration. Therefore, the stability of the two CKM and the model-drug insulin towards collected porcine gastric and small intestinal juice as well as towards isolated proteolytic enzymes was evaluated under physiological conditions. No intact SE-ET-EP-020 was detected after few seconds of incubation with porcine small intestinal juice. SE-ET-TP-020 was also degraded in porcine gastric juice. Furthermore, SE-ET-TP-020 was extensively degraded by isolated chymotrypsin, trypsin and pepsin. Moreover, it was degraded by elastase. SE-MC-TR-020 was degraded entirely within approximately 2 h when incubated in porcine small intestinal juice, whereas no degradation was observed within a 3 h incubation period with porcine gastric juice. In presence of the isolated proteolytic enzymes, SE-MC-TR-020 was only slightly degraded by trypsin and pepsin, whereas elastase caused no degradation to SE-MC-TR-020 at all. Chymotrypsin was the protease that caused most degradation to SE-MC-TR-020. The model drug insulin was degraded extensively by chymotrypsin, elastase, pepsin and trypsin as well as by porcine gastric and porcine small intestinal juice. In conclusion, a precise characterisation of SE-ET-TP-020 and SE-MC-TR-020 degrading luminally secreted GI enzymes has been made, which is an important and substantial prerequisite for the further optimisation of these CKM.     

Isolation and partial characterization of prochymosin and chymosin from cat

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 cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general 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.