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The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.

Technology for preparation of chymosin from milk of transgenic sheep has been elaborated. Purification of the preparation by ion-exchange chromatography on aminosilochrom and biospecific chromatography on bacitracin-Sepharose yielded homogeneous active enzyme. Hydrolysis of protein substrates (hemoglobin, BSA, and sodium caseinate) by the transgenic sheep chymosin and stability of the enzyme at various values of pH were studied. Judging by the amino acid composition, the N-terminal sequence involving six amino acid residues, molecular mass, stability at various pH values, and the catalytic activity against the protein substrates, the transgenic sheep chymosin is identical to calf chymosin.

The use of fluorogenic substrates with intramolecular fluorescence quenching as substrates for chymosin was studied. It was shown that chymosin hydrolyzes the Phe-Phe peptide bond. The effect of pH on the hydrolysis of substrates by chymosin was investigated. The catalytic characteristics of the hydrolysis of the fluorogenic substrates were obtained at the pH optima. The influence of dimethylformamide on chymosin activity was studied.

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.

Buffalo chymosin was isolated from abomasum mucosa extract of buffalo calves by affinity chromatography on gramicidin S-agarose followed by ion exchange chromatography on gamma-aminopropylsilochrom. Its molecular weight, 36 +/- 1 kDa, is similar to that of bovine calf chymosin. The N-terminal sequence Gly-Glu-Val-Ala-Ser-Val-Pro- coincides with that of bovine enzyme, whereas some differences were found in the amino acid composition of these enzymes. Buffalo and bovine enzyme possess similar but not identical structures. General proteolytic and milk-clotting activities of buffalo chymosin are also similar to those of bovine proteinase. pH-Optimum of its activity against hemoglobin lies at pH 4.0, somewhat higher than that for bovine chymosin, which indicates subtle differences in the functional properties of two enzymes.

Porcine pepsin behaviour during the synthesis of peptide p-nitroanilides and esters has been studied. In many cases, especially when long-chain peptides, such as Z-Ala-Ala-Phe-Leu-Ala-Ala-OMe, were synthesized, pepsin disappeared from the solution, being entrapped by the product precipitate rather than inactivated. Sorption of the enzyme on the product might be partially responsible for this effect. The active enzyme could be eluted from the precipitate by NaCl and isopropanol. Non-proteolytic proteins (lysozyme, bovine albumin, carbonic anhydrase) could also co-precipitate with pepsin.

Swine pepsin at pH 5 efficiently catalyzes a condensation between Z-Ala-Ala-Phe-OH and p-nitroanilides of Leu, Phe, Val, Ala and Arg that leads to formation of corresponding benzyloxycarbonyl-tetrapeptide p-nitroanilides with yields of 70-90%. These reactions are complicated by co-precipitation of pepsin and the reaction products that necessitates the use of a relatively high concentration of pepsin.

Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri-, tetra-, penta- and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide-water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved--up to 1:3.10(5) for relatively long peptides.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.

Metalloproteinase of Legionella pneumophila is the major extracellular proteinase of this bacterial species which splits human immunoglobulin G in the hinge region to form the (Fab')2 fragment. This fragment is relatively stable and undergoes further proteolysis at a slow rate. The c' fragment is unstable and is apparently split down to fragments CH2 and CH3. The metalloproteinase splits human serum albumin down to products having lower molecular masses. Another bacterial metalloproteinase, thermolysin, produces a similar effect, although at a slower rate.

Antitryptic activity of human blood serum was decreased after incubation with metalloproteinase from Legionella pneumophila. The enzymatic activity depends on the time of incubation as well as on the ratio between the enzyme content and blood serum total protein. Cross immunoelectrophoresis, involving monospecific rabbit antiserum towards the alpha 1-antitrypsin, demonstrated highly effective hydrolysis of alpha 1-antitrypsin by the metalloproteinase. As shown by polyacrylamide gel electrophoresis the metalloproteinase hydrolyzed acid stable inhibitor of serine proteinases into fragments with distinct loss of the inhibitor activity. Thermolysine hydrolyzed similarly the proteins studied but at a lower rate. The metalloproteinase from L. pneumophila appears to be mainly responsible for production of utilizable components from protein substrates involved in vital activity of the bacteria. It may not be excluded that the enzyme is able to impair some host protective mechanisms.

Calf chymosin catalyzes peptide synthesis optimally at pH 4-5 giving satisfactory yields of methyl esters or p-nitroanilides of benzyloxycarbonyl tetra- to hexapeptides, provided that hydrophobic amino acid residues form the new peptide bond. The enzyme efficiency depends also on the nature of adjacent amino acid residues. As an aspartyl proteinase with characteristic specificity pattern chymosin would be useful for synthesis of middle length peptides.

It has been shown that in the course of equilibrium peptide synthesis pepsin gradually disappeared from the liquid phase due to its entrapment within a gel formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin so far as inert proteins-lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin-an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.

Prochymosin can be converted into chymosin by an action of external proteinases. Thus, thermolysin at pH 5.05 converts calf prochymosin into active Phe-chymosin, which is one amino acid longer than chymosin from the N-terminus with a yield of 73%. Even better results were achieved with prochymosin activation by Legionella pneumophila metalloproteinase. Apparently the stretch of prochymosin polypeptide chain adjacent to the normally observed activation point becomes available for an attack by an external proteinase at pH 5.0-6.0. These data indicate that the intermolecular activation pathway might be of physiological importance.

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.

Using ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on DNP-hexamethylenediamine-Sepharose and gramicidin S-Sepharose and gel filtration, a metalloproteinase was isolated from the cultural fluid of L. pneumophila (strain Philadelphia-1) grown for 20 hours. The enzyme was purified 1606-fold with a 31% yield. The enzyme has a Mr of 38,000, pI approximately 4.0 and optimum of proteolytic activity at pH 6.0-7.0, 55 degrees C. The proteinase is the most stable within the pH range of 6.0-9.0. The enzyme contains one atom of zinc per molecule. The amino acid composition of metalloproteinase is close to that of thermolysin and is characterized by a high methionine content--17 residues out of 348. In the B-chain of oxidized bovine insulin the enzyme hydrolyzes the bonds precedent to the amino groups of leucine, phenylalanine and tyrosine. The enzyme is inhibited by chelating agents--Na2-EDTA and o-phenanthroline as well as by diethylpyrocarbonate. The serine and thiol proteinase inhibitors do not influence the enzyme activity. Under the given conditions of cultivation metalloproteinase is the major endopeptidase produced by L. pneumophila. Thus, the proteolytic system of Legionelles is characterized by the combination of metalloproteinase and the earlier described phenylalanine aminopeptidase.

6 forms of pepsin are found in horse gastric juice. Amino acid sequence is determined of N-terminal (most variable) part of polypeptide chain of main pepsin components. Equine pepsines, which have pI 2.1 and 2.3, are found to have identical amino acid sequence at least for 31 amino acid residues. The same sequence is observed in the component with pI 2.6 for 10 first residues. The sequence of equine pepsin with pI 3.2 has 3 substitutions for 33 amino acids, when compared with pepsines having pI 2.1 and 2.3. The forms of equine pepsin studied are more similar than the other isoenzyme pair, human pepsin and gastricsin.

Serine proteinase and metalloproteinase of Asp. oryzae, extracellular metalloproteinase of L. pneumophila and chymotrypsin-like proteinase of S. rutgersensis can hydrolyze pepsinogen by converting it into pepsin (pH 5.0, 37 degrees C). The localization of the site of hydrolysis depends on the nature of the enzyme: serine proteinase from Asp. oryzae induces the synthesis of a mixture of 60% pepsin, 25% leucyl-pepsin and 15% alanyl-leucyl-pepsin; metalloproteinase of Asp. oryzae converts pepsinogen only into leucyl-pepsin, while metalloproteinase of L. pneumophila yields a mixture of 33% pepsin, 53% leucyl-pepsin and 14% alanyl-leucyl-pepsin. Thus, the region of the activating pepsinogen peptide--Ala 42P-Ile 1 bond--seems to the most probable site for hydrolysis by exogenous proteinases. This site contains a Leu 44P-Ile 1 bond which is subjected to intermolecular hydrolysis during autocatalytic activation of pepsinogen. The experimental results emphasize the importance of the intermolecular pathway of pepsinogen activation.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.

The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.

Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilo chrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards L-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.

Extracellular serine proteinase of Streptomyces rutgersensis (proteinase A) has been isolated by affinity chromatography on bacitracin-Sepharose in the presence of Na2EDTA. The proteolytic activity of the enzyme is completely suppressed by specific inhibitors of serine proteinases, phenylmethylsulfonylfluoride and diisopropylfluorophosphate, but is insensitive to Na2EDTA and sodium p-hydroxymercuribenzoate. The enzyme activity towards hemoglobin is maximal at pH 9-10, the maximal stability is observed within the pH range of 6-10. The molecular mass as determined by SDS-pore gradient electrophoresis is equal to 20000 +/- 2000. Proteinase A is similar to the proteinases A and B of Streptomyces griseus in terms of i) amino acid composition--Asp17Thr22Ser23Clu7Pro6. X Gly30Ala13Cys4Val18Met1Ile5Leu9Tyr10Phe6Trp1His2Arg7, ii) amino terminal sequence--Leu-Ser-Gly-Gly-Asp-Ala-Ile-Tyr-Ser-Asn-Ser-Ser-Xaa-Xaa-Ser-Leu-, iii) specificity of insulin B-chain hydrolysis, and iiii) ability to exhibit proteolytic activity in 8 M urea or 6 M guanidinium chloride. The enzyme lyzes the cells of Candida utilis. Besides proteinase A, S. rutgersensis produces at least two other proteolytic enzymes.

Pepsin chromatography was studied on peptide ligand sorbents, differing in the length of the polypeptide chains, ionogenic groups and the nature of hydrophobic side groups. Pepsin sorption was found to be dependent of a specific interaction of the substrate analogs with the enzyme and ionic interactions with the matrix and ionogenic groups of the ligand. Non-specific hydrophobic interactions of the enzyme and the ligand have little effect on the sorption. Efficient methods for the isolation of pepsin and separation of the mixture of bovine chymosin and pepsin are described.

A sorbent obtained by treatment of cyanogen bromide-activated Sepharose 4B with mono-N-DNP-hexamethylenediamine has been shown to be effective in the affinity chromatography of pepsin, pepsinogen and acid proteinase from Aspergillus awamori. It is considered that 2,4-dinitrophenyl residues of the sorbent interact specifically with the hydrophobic zone of the enzyme, which may belong to the substrate binding site. The chromatography of chymotrypsin on the same sorbent supports this assumption.

It was found that at pH 5.2 and 40-fold excess of p-nitrophenyldiazonium chloride the inhibitor incorporation into the porcine pepsin molecule involves 1.9 residues, one residue being bound to tyrosine 189. Besides, tyrosines 44, 113, 154 and 174 enter the reaction. Modified pepsin retains 25% of the native enzyme activity. In the pepsinogen molecule the degree of tyrosine 189 modification diminishes 5 times; of 1.5 inhibitor molecules incorporated into the protein 0.78 residues are bound to tyrosine 113. The potential proteolytic activity of modified pepsinogen towards haemoglobin cleavage makes up to 60% of the original one. It is concluded that the activation peptide in the pepsinogen molecule masks the substrate binding site bearing tyrosine 189, thus preventing its modification with p-nitrophenyldiazonium chloride. The activation peptide in the pepsinogen molecule is presumably located in the vicinity of the wide loop bend carrying tyrosine residue 113, which may be the reason for the decreased pKa value of this residue and of its increased reactivity in the azocoupling reaction.