Hydrolysis of Irradiated Ovalbumin by Pepsin

Solid ovalbumin has been irradiated at three doses, 6.5, 25, and 40 megarads, under high vacuum. The native and irradiated samples have been hydrolyzed at pH 1.4 by pepsin, after centrifugation of the aggregates, if necessary. The number of bonds broken per ovalbumin molecule has been estimated by comparing the rate of protein destruction with the rate of formation of NH₂ groups. Both rates increase very much with the dose, but the number of bonds broken decreases. Sedimentation measurements show a strong shape modification of the soluble fraction in the case of the 25 and 40 megarad samples. The increase in asymmetry is bound to the increase in the rate of attack on γ-irradiated ovalbumin by pepsin. Infrared spectra of the aggregates show small difference from those of the native samples.     

Improved pepsin inhibitor derived from activation peptide 1-16 of porcine pepsinogen.

The peptide Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-Ser-Leu-Arg-Gln-Asn-Leu, a known pepsin inhibitor, is derived from the first 16 amino acids of porcine pepsinogen. It was prepared from the activation mixture and was modified by guanidination of its three lysine residues to form homoarginine residues. The modified peptide is a better pepsin inhibitor than the native peptide; for 50% inhibition of the milk clotting action of pepsin at pH 5.3, the molar ratio of peptide to pepsin required is 9 for the native inhibitor and only 2 for the guanidinated inhibitor. The dissociation constants (k1) of the inhibitor-pepsin complexes are 7 X 10(-8) and 1.4 X 10(-8) M for the native and guanidinated peptides, respectively. The guanidinated peptide is more resistant to digestion by pepsin at pH 3.5. The native and modified peptides partially protect pepsin from inactivation at pH 7. Stepwise removal of the amino-terminal Leu-Val-Har residues from the guanidinated inhibitor by Edman degradation decreases the pepsin-inhibiting activity only slightly at the first step, but markedly at the second and third steps. Thus, all of the amino-terminal sequence except the leucine residue is necessary for full activity.     

ACETYLATION OF TYROSINE IN PEPSIN

Crystalline 60 per cent active acetyl pepsin has 7 acetyl groups per mol of pepsin, 3 of which are readily hydrolyzed in acid at pH 0.0 or in weak alkali at pH 10.0. The tyrosine-tryptophane content of this acetylated pepsin, measured colorimetrically, is less than pepsin by three tyrosine equivalents. Hydrolysis at pH 0.0 or pH 10.0 of the 3 acetyl groups results in a concomitant increase in the number of tyrosine equivalents. In the pH 0.0 hydrolysis experiment there is also a simultaneous increase in specific activity. The phenol group of glycyl tyrosine is acetylated by ketene under the conditions used in the acetylation of pepsin and the effect of pH on the rate of acetylation is similar in the two cases. It is concluded that the acetyl groups in the 60 per cent active acetyl pepsin, which are responsible for the decrease in specific enzymatic activity, are 3 in number and are attached to 3 tyrosine phenol groups of the pepsin molecule.     

The pepsins of normal human gastric juice

1. The frequency of occurrence, under defined conditions, of the different human pepsins in the gastric juices of 50 normal subjects was investigated by agar-gel electrophoresis. 2. From a total of eight proteolytic zones located in the zymograms, no significant differences of occurrence existed between the sexes, or between subjects with or without gastric symptoms. 3. Two zones, numbered 3 and 5, occurred in all normal gastric juices. Zone 3 always exhibited the greatest proteolytic activity, then zone 5. The remaining enzymic zones were less well-marked and occurred less frequently. 4. A minor zone, 3a, was demonstrated within zone 3. The corresponding pepsin, 3a, has a mobility towards the anode 6-7% greater than has pepsin 3. 5. Of the eight zones, 1,2,3,3a and 5, at least, represent unique pepsins.     

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.     

Re-formation of disulphide bonds in reduced antithrombin III.

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.     

The specificity of some pig and human pepsins towards synthetic peptide substrates.

1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin.     

Detection and measurement by high-performance liquid chromatography of malondialdehyde crosslinks in DNA

Val-D-Leu-Pro-Phe-Phe-Val-D-Leu, a specific inhibitor of aspartate proteinases of the pepsin type, was synthesized. Its bonding to activated 6-aminohexanoic acid-Sepharose 4B afforded an affinity support suitable for the purification of human, porcine, and chicken pepsin, human gastricsin, and bovine cathepsin D. These enzymes bind to the support over the pH range 2-5 at 0-1.5 M concentration of NaCl. A buffer at pH greater than or equal to 6, low ionic strength, and containing 20% dioxane can serve as a general desorption agent. The proteinases were isolated from the crude extracts by a single-step procedure in a high degree of purity and in yields exceeding 70%; human pepsin, however, was not separated from human gastricsin. The support does not show any binding capacity for rat plasma renin at pH 7.4 and for some cysteine endopeptidases (cathepsin B, H, and L) at pH 3-5. The cathepsin D preparations isolated by affinity chromatography on the new support and on pepstatin-Sepharose were of the same degree of purity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequences, and specific activity.     

Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin

In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, K_m and V_max were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (K_m = 15 g/L, V_max = 719 U/mg versus K_m = 12.6 g/L and V_max = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (K_m = 18 g/L, V_max = 685 U/mg). The values for the anchored enzyme-glass were K_m = 19 g/L, V_max = 763 U/mg.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

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.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography.

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.     

Activation of proenzyme of acidic protease from human seminal plasma.

The kinetics and the extent of the conversion of the proenzyme into the active acidic protease (EC 3.4.23.--) of human seminal plasma were dependent on acidic pH. Between pH 2 and 4, the initial rate of the activation was first-order with respect to the proenzyme. Between pH 4.5 and 5, the rate deviated from the first-order with an initial lag period which can be abolished by adding an excess amount of the acidic protease or pepsin. The extent of the activation was complete between pH 2 and 3 and became incomplete between pH 4 and 5. Addition of the acidic protease or pepsin did not alter the extent of the activation at the high pH values. According to the chromatographic profile on a Sephadex G-75 column, the activation products (namely active acidic protease and an activation peptide) obtained at pH 3 and those obtained at pH 4.5 were identical. The molecular weight of the activation peptide obtained at pH 3 was 6900; its amino acid composition was analyzed and compared with those of the proenzyme and the acidic protease. Remarkable similarity between the amino acid composition of the acidic protease and that of human pepsin was observed. In the presence of an excess amount of hemoglobin, the conversion of the proenzyme was self-activated and showed an initial lag period. Addition of acidic protease did not change the rate of self activation or the lag period.     

Inhibition of human pepsin and gastricsin by alpha2-macroglobulin

The inhibitory effects of human alpha2-macroglobulin (alpha2-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by alpha2-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved alpha2-M mainly at the His694-Ala695 bond and Leu697-Val698 bond, respectively, in the bait regions sequence of alpha2-M. The conformation of alpha2-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by alpha2-M.     

Properties of the activation by pepsin of inactive renin in human amniotic fluid.

1. The renin present in human amniotic fluid was found to have an apparent Mr of 58 000 by gel filtration and is thus bigger than renin in untreated kidney extracts and plasma (Mr approximately 40 000). 2. Treatment with pepsin (40 microgram/ml pH 4.8, 2 h, 22 degrees C) caused a 6-fold increase in activity of this renin species, although Mr was not very different (57 000). 3. Unlike renal renin, renin in human amniotic fluid was not a glycoprotein and behaved similarly on concanavalin A-Sepharose before and after activation by pepsin. 4. Ion-exchange chromatography demonstrated a small change in the ionization properties of human amniotic fluid renin after activation by pepsin. 5. Pepsin-mediated activation resulted in a five-fold increase in V, but only a small decrease in the Km of renin to 39% of normal, so that the increase in activity observed was not due to an increase in the affinity of the enzyme for its substrate. The kinetic data were consistent with the theory of noncompetitive inhibition. 6. The activation of human amniotic fluid renin by pepsin may be caused by a change in the tertiary structure of the molecule subsequent to a proteolytic action that does not remove detectable polypeptide components.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

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.     

Comparison of pepsins isolated from porcine, bovine and Penicillium jathiuellum

1. The reactivities of pepsins, isolated from three different sources (porcine, bovine, and Penicillium jathiuelum), toward ester and peptide substrates were compared. 2. Porcine pepsin showed the highest activity followed by penicillopepsin with bovine pepsin being the least active. 3. The esterase activity of penicillopepsin was greater than that of porcine pepsin with bovine pepsin again showing the least activity. 4. The CD spectra indicate that porcine and bovine pepsin have similar conformations, even though bovine pepsin shows less ellipticity at 220 nm. 5. Penicillopepsin showed a completely opposite sign in the near-u.v. region of the CD spectrum. 6. The far-u.v. region of the CD spectrum of penicillopepsin strongly suggests a beta-sheet structure. 7. Previously reported X-ray crystallographic data suggest that porcine pepsin has a compact three-dimensional structure, while the structures of bovine and penicillopepsins are partially unfolded.     

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.