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An anionic chymotrypsin-like enzyme was isolated from a crude extract of camel pancreas by a three-step procedure consisting of anion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was homogeneous on native and SDS gel electrophoresis and on gel isoelectric focusing. Its molecular mass was estimated as 28.5 kDa and its isoelectric point was found to be 4.4. The enzyme differed markedly from bovine chymotrypsin A in its substrate specificity, showing considerably lower values of the specificity constant for its action on tyrosine, tryptophan, and phenylalanine esters. Its pH optimum was found to be 7.8. It showed lower kininase activity and was more susceptible to inhibition by a number of inhibitors than the bovine cationic chymotrypsin. On the other hand, the camel enzyme showed a much greater hydrolytic activity than the bovine enzyme toward a leucine ester. In terms of its size, charge, and substrate specificity the camel enzyme was very similar to anionic chymotrypsins that have been isolated from other species and thus appears to be a camel anionic chymotrypsin.

Previous investigation [Tsui et al. (1996) Biochim. Biophys. Acta 1269: 41-46] showed that two active forms of alcohol dehydrogenase can be purified from grass carp. The use of a protease inhibitor and the results of SDS-PAGE analysis of the enzymes suggest that one form (ADH-C) is a proteolytic product of the other (ADH-I). In this study, the protease responsible for the cleavage was purified. The cleavage enzyme had a subunit molecular weight of 28 kDa. An inhibitor study identified it as a serine protease. It exhibited a strong chymotrypsin activity in both esterase and amidase assays with a pH optimum in the range 7.5-8.5. The purified chymotrypsin also cleaved the intact grass carp ADH-I into the two-fragment ADH-C, with an accompanying increase in enzyme activity. A similar effect was not found using horse liver alcohol dehydrogenase.

A novel thermostable protein inhibitor of trypsin and subtilisin, called BN, was isolated from the seeds of Brassica nigra. The purified protein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500 +/- 1000 Da. The inhibitor is composed of two disulfide-linked polypeptide chains, consisting of 39 and 90 residues, respectively. The amino acid sequence of the two chains was determined by Edman degradation of peptides, isolated from enzyme hydrolysates with TPCK-trypsin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples. A segment of the 'heavy' chain, between residues 65 and 81, showed homology with the reactive site loop region of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 (N. alata) or 70 (napins) is conserved as arginine or lysine in all inhibitors from N. alata and in all napins hitherto sequenced. Probably, the two families of trypsin inhibitors have structurally similar reactive sites. BN exhibits an extremely high thermostability: CD measurements showed that during heating to 97 degrees C it preserves a considerable part of the polypeptide backbone folding. Studies on the fluorescence properties of the inhibitor BN in the absence and presence of neutral or ionic quenchers demonstrated that the intrinsic emission of this protein is dominated by a tryptophyl residue, buried in the interior of the protein matrix. 20% of the light absorbed by Tyr 63 of the 'heavy' chain is transferred to Trp 26 of the 'light' chain.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin were prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.

A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.

Background: The allergen Der p 6 from Dermatophagoides pteronyssinus has been described by substrate affinity as mite chymotrypsin., Objective: The aim of this paper was to describe a cDNA clone encoding the allergen., Methods: cDNA was cloned from a lambda library using oligonucleotides published for Der p 6., Results: The clone P6.1.1 had the N-terminal amino acid residues as reported for Der p 6, and at position 189 the Ser was conserved in chymotrypsin for substrate specificity as well as the catalytic triad of His57, Asp102 and Ser195 for serine proteases. The estimated M(r) was 24.9K and it was 37% identical to the trypsin allergen Der p 3., Conclusion: cDNA encoding Der p 6 has been described.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

The molecular weights of trypsin and chymotrypsin purified from anchovy viscera were estimated to be 25.6 and 26.1 Kda, respectively, by SDS-PAGE. Both enzymes had their maximal activity at pH 9.0 and 45 degrees C for casein and at pH 8.0 and 45 degrees C for synthetic substrates. Trypsin hydrolyzed at the position of Arg22 and Lys29, and chymotrypsin did at the position of Phe1, Tyr16, Phe24, Phe25, and Tyr26 of insulin beta-chain. The K'm and kcat of trypsin were 50 microM and 1.84 microM-1 min-1 toward N-benzoyl-L-arginine-p-nitroanilide (BAPNA) and those of chymotrypsin were 89 microM and 10.0 microM-1min-1 toward N-succinyl-(Ala)2-Pro-Phe-p-nitroanilide. The activation energy of trypsin and chymotrypsin were estimated to be 14 Kcal/mol toward N-benzoyl-L-arginine-p-nitroanilide and 6.5 Kcal/mol toward benzoyl-L-tyrosine ethyl ester.

The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.

The extraction of lysozyme, alpha-chymotrypsin, and pepsin from buffered salt solutions into reverse micelles was examined at different pH values and surfactant concentrations. The reverse micelles was formed by mixing aqueous buffer supplemented with KCl and an organic phase of isooctane(2,2,4-trimethylpentane), containing the anionic surfactant, Aerosol O. T. (dioctyl ester of sodium sulfosuccinic acid). The technique of dynamic laser scattering was used to measure the size of reverse micelles which were in equilibrium with the aqueous phase. It was found that the size of the reverse micelles decreased with increasing ionic strength but increased with increasing AOT concentration. In the process of extraction, the reverse micelles might have rearranged themselves to host the protein. The sizes of protein-filled and -unfilled reverse micelles were different, and an open equilibrium could be reached between them. Under the extraction conditions, only a small number of micelles were found to contain protein.

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

The proteolytic activities of extracts prepared from procercoids and plerocercoids as well as adults of the pseudophyllidean cestode Schistocephalus solidus were examined using several proteins and synthetic peptides as substrates. Whole bodies of procercoids and the isolated syncytial tegument of plerocercoids and adults prepared by freeze-thaw were studied. Extracts of procercoids contained a chymotrypsin-like proteinase exhibiting a molecular weight of 23,500 determined by gel filtration chromatography. The proteinase showed collagenolytic activity and had a pH optimum at 8. Such a proteinase was absent in plerocercoids and adults. In these developmental stages leucine aminopeptidases were detected in the isolated syncytial tegument having molecular weights of 93,500 (plerocercoids) and 89,000 (adults), respectively. The aminopeptidases in both stages displayed optimal activity at pH 8.5. The chymotrypsin-like proteinase of the procercoid is possibly necessary for the penetration of the host's intestinal wall, whereas the aminopeptidases of the plerocercoid and the adult of S. solidus may aid in parasite nutrition by degrading oligopeptides at the tegumental surface.

A clone expressing a Treponema denticola chymotrypsinlike protease from recombinant plasmid pSA2 was identified in a genomic library of T. denticola ATCC 35405. Nucleotide sequencing of the insert identified an open reading frame, designated the prtB gene, which codes for the protease. Two potential inverted repeat sequences are present both upstream and downstream from the prtB gene. The prtB gene would code for a putative protein of 273 amino acids with a calculated molecular mass of 30.4 kDa and an estimated pI of 7.0. The G+C content of the gene is 40.3%. The results of maxicell analysis are consistent with the expression of a 30-kDa protease from the prtB gene. Preliminary characterization of the protease indicated that it was inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and N-tosyl-L-phenylalanine chloromethyl ketone but not by N alpha-p-tosyl-L-lysine chloromethyl ketone. Purification of the protease was accomplished with the PinPoint protein purification system following construction of site-directed mutagenized plasmid pXa-3:2. The purified protease degraded human and bovine serum albumins as well as casein. Furthermore, hemolysis of sheep erythrocytes by the protease was observed. Northern (RNA) blot analysis of mRNA extracted from strain 35405 indicated a single 1.9-kb mRNA species containing the prtB transcript. In addition, the results of primer extension analysis indicated that transcription was initiated primarily at a T residue. However, no corresponding -10 and -35 sequences related to Escherichia coli promoter sequences were identified. The availability of the purified protein and its gene will aid in evaluating the potential role of the protease in the physiology and virulence of T. denticola since proteases may play a key role in oral treponemal pathogenicity.

We have obtained unusual 'zig-zag' temperature dependencies of the rate constant of irreversible thermoinactivation (k(in)) of enzymes (alpha-chymotrypsin, covalently modified alpha-chymotrypsin, and ribonuclease) in a plot of log k(in) versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy (Ea), respectively. A kinetic scheme has been suggested that fits best for a description of these zig-zag dependencies. A key element of this scheme is the temperature-dependent reversible conformational transition of enzyme from the 'low-temperature' native state to a 'high-temperature' denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low-temperature and high-temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of delta H and delta S for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperature conformation, alpha-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.

The mast-cell-specific proteolytic enzymes tryptase and chymase were identified in and isolated from cholesteatoma in a ratio similar to that found in human skin. We assume that this ratio reflects a similar distribution of tryptase-containing and tryptase/chymase-containing mast cells in both these tissues. It seems conceivable that mechanisms able to trigger excessive and/or continuous mast cell degranulation in the middle ear might be causative for the formation of cholesteatoma either directly or via primed chronic inflammatory reactions. By their ability to amplify degranulation of mast cells, mast cell proteinases, in particular chymase, may contribute to the chain of events leading to the formation of cholesteatoma.

The results of an electrospray-mass spectrometric analytical study of aqueous solutions of fifteen commercial samples of 3X-recrystallized bovine alpha-chymotrypsin are presented and discussed. It was found that only six samples were predominantly alpha-chymotrypsin and that two samples contained no alpha-chymotrypsin at all. The remaining seven samples were found to be mixtures of alpha-chymotrypsin with other chymotrypsins and, in some cases, neochymotrypsinogens. The majority of the results are rationalised in terms of previously postulated and/or observed products of proteolytic activation of bovine chymotrypsinogen A. However, evidence is also presented for the presence in many of the samples of three new serine proteases, of significantly lower molecular masses than alpha-chymotrypsin, which cannot at present be explained. The paper is concluded with a brief discussion of the implications of the analytical findings for enzymological studies.

We have purified two chymotrypsin-like proteases from chum salmon sperm which have no apparent acrosome structure. Both of them were high molecular mass proteases (650 kDa and 950 kDa by gel filtration) and showed not only chymotrypsin-like activity but also trypsin-like activity. The 650 kDa protease was composed of at least eight or nine kinds of polypeptide with molecular masses ranging from 20 kDa to 30 kDa and was highly activated by low concentrations of SDS. Electron microscopy revealed that the 650 kDa protease was a ring-shaped particle. The 950 kDa protease was shown to contain at least one component that cross-reacts with an antibody against the 650 kDa protease. Finally, we revealed that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-activation and molecular shape of 650 kDa salmonid protease were quite similar to those of the eukaryotic multicatalytic proteinase (proteasome), which is well known to participate in ATP-dependent degradation of ubiquitinated proteins; and, furthermore, the motility of demembranated sperm of salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. Thus, the protease located in salmonid fish sperm flagella is a proteasome and is a strong candidate for the factor which regulates flagellar motility in an ATP-dependent manner.

1. Two chymotrypsin variants, with collagenolytic activities, were purified from the hepatopancreas of Penaeus vannamei using radioactive protein as the substrate. 2. These proteases are very close as far as amino acid composition, molecular weight, inhibitors studies and specificity against small synthetic substrates are concerned. 3. N-terminal amino acid sequences of both variants are identical and are very close to other known crustacean serine proteases.

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.

1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+/- 1200) and 28,800 (+/- 900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55 degrees C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine alpha-chymotrypsin when assayed against Suc-AAPF-NA at 25 degrees C and casein at low temperatures (10-20 degrees C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.

Genetic variation of a codominantly inherited pancreas protease, designated CTRA-1, was discovered in the house mouse by isoelectric focusing in polyacrylamide gels. Phenotype CTRA-1A was found in MOLH/Fre and in the majority of common laboratory mouse strains. Phenotype CTRA-1B was found in PWD/Ph. It was characterized by the absence of a corresponding protease band. A third phenotype, CTRA-1C, was observed in IS/Cam and a fourth phenotype, CTRA-1D, was detected in SEG/1. CTRA-1 was found only in the pancreas and may represent the A form of chymotrypsin. The enzyme was shown to be controlled by the presumed structural locus Ctra-1 located on chromosome 8. From two backcross series, including a total of 274 animals, the gene order (Es-1, Es-9)-3.9 +/- 1.7%-Got-2-3.9 +/- 1.7%-(Es-2, Es-7, Es-23)-0.7 +/- 0.5%- Ctra-1-6.3 +/- 2.2%-Prt-2 was established.

The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.

The authors treated destructive forms of acute pancreatitis for the first time by means of specific plasma sorption of proteinases with the use of a proteinase plasma sorbent which is an acid-stable proteinase inhibitor immobilized on sepharose (ASI-Sepharose). Specific plasma sorption of proteinases on ASI-Sepharose was applied to the treatment of 29 patients with various forms of acute destructive pancreatitis. At the end of specific plasma sorption procedure the activity of blood plasma proteolytic enzymes in the patients reduced by 60-75%, which was attended by marked improvement of the condition in most cases; aggravation of the patients' condition was not encountered. The mortality in this group was 20.7% and was due to complications occurring during the development of the main disease. The mortality rate among patients treated by specific proteinase plasma sorption is much lower than that recorded in the USSR for destructive forms of acute pancreatitis which ranges from 30 to 75%.

The mass and activity recovery of eight different enzymes (two monomeric, six oligomeric) with molecular masses between 25,000 and 240,000 daltons were tested after HPLC separation on three different HPLC instruments (two with stainless steel and one with titanium flow paths). Most of the tested proteins are known to be sensitive to heavy metal ions. Eight wide pore, ion-exchange columns, two size-exclusion columns and two hydrophobic-interaction columns were used. Both stainless steel and glass column hardware were used in all three separation modes. The elution times were between 8 and 12 minutes. In almost all cases, the activity recovery was between 90% and 100% compared with a control sample incubated in the chromatographic elution buffer for the same time at the same temperature. A severe activity loss (about 30%) was observed with only one ion-exchange column and one enzyme. Neither the column hardware nor the material of the HPLC equipment had any negative effect on the activity recovery of the enzymes tested.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.

Aprotinin isolated from bovine lungs was covalently immobilized on Eupergit C. The highly selective affinity adsorbent was used to purify trypsin and alpha-chymotrypsin from pancreatic extract in a single step. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that both enzymes were highly purified. The maximum binding capacity of Eupergit-aprotinin for bovine trypsin was calculated to be ca. 7.5 mg per gram of gel (wet resin).

A chymotrypsin-like proteinase was purified 2400-fold from human skin. The procedure involves extraction of the proteinase from skin in 2 M KCl, precipitation with protamine chloride, fractionation by gel filtration chromatography, and fractionation by chromatography using a CH-Sepharose-D-tryptophan methyl ester affinity column. The properties of this proteinase were compared to the rat mast cell proteinase I and human cathepsin G. Differences were observed in the rates at which the proteinases were inhibited by diisopropyl fluorophosphate, the sensitivity of the proteinases to protein proteolytic inhibitors, the relative hydrolytic rates of the proteinases for a series of substrates, and the kinetic constants of the proteinases for synthetic substrates. The human skin proteinase did not react with antiserum to the rat skin proteinase and did not elute in the same position as the rat skin proteinase on gel filtration columns. These data demonstrate that the human skin proteinase is distinct from the other proteinases. Extracts of involved skin from patients with cutaneous mastocytosis had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or skin from normal controls. The enzymatic properties of the material extracted from the biopsied skin were similar to those of the proteinase from normal skin, suggesting that the human skin chymotrypsin-like proteinase is a mast cell constituent.

A study in the properties of hydrolytic enzymes and the processes of hydrolysis relized by them is a main trend in the department activity. Special attention is paid to the studies in hydrolases of microbial origin. Methods are developed for obtaining the proteolytic and cellulolytic complexes synthetized by the Aspergilla and Actinomyces. New, nonstudied (or little studied) individual enzymes are isolated from the proteolytic complexes proteinases form Asp. flavus, Asp. oryzae, Str. griseus; carboxypeptidase from Str. griseus. LGG-aminopeptidase from Asp. flavus. The presence of dipeptidases of different types is shown. The methods for cystallization of pepsin, alpha-chemotrypsin, trypsin, protease from Str. griseus, alpha-amylase from Asp. cryzae are developed and improved. The properties of the isolated enzymes are studied--their substrate specifity, elements, of chemical structure, effect of activators and inhibitors, metal ions, etc. Special attention is paid to studies in stability and conditions of enzymic proteins stabilization. On the basis of studies in the field of preparative enzymology as well as in stabilization and denaturation of the enzymes, seven preparations (or new methods of their production) are worked out for application to medicine. The studies in the process of gelatin hydrolysis with protease of Str. griseus made it possible to develop a new technique for silve regeneration by means of the preparation "proteinase-1". The enzymic-antibiotic preparation "protezym" proved to be effective when feeding sucking pigs and broilers.

Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively eluted either with acetic acid or with 40% glycerol, pH 8.0. This is the first report of a preparative separation of free and proteinase-bound alpha 2-macroglobulin. Using this affinity chromatographic system, we were able to demonstrate that the two chymotrypsin binding sites of alpha-2-macroglobulin are equivalent and independent.

The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F-, Cl-, SCN-, ClO-4, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F-, Cl-, SCN-, and ClO-4, and low (less than 0.003 M) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F-, but do not elute with Ca2+ and usually not with Cl-; (3) neutral proteins, which elute with PO4, F-, and Cl-, but show a strong anion specificity, and do not elute with Ca2+ or SCN-. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.

Malignant and uninvolved human breast tissues were maintained in organ culture for 3 to 6 days. Under these conditions, the three-dimensional glandular architecture is maintained with the least disruption of tissue integrity. The biosynthesis and release of glycoproteins were studied by using the incorporation of [14C]glucosamine and [14C]leucine by the breast surgical specimens. Five major families of labeled glycoproteins were identified from culture supernatants using two-dimensional gel electrophoresis. Quantitative immunoprecipitation established that 16 to 30% of the total of labeled glycoproteins were recognized as normal serum components. Two of these glycoproteins were antigenically related to normal human serum components as demonstrated with crossed immunoelectrophoresis. Evidence was obtained for the active synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. alpha 1-Antichymotrypsin accounted for 0.9 to 7.8% of the biosynthetically labeled glycoproteins from organ culture supernatants. This component was 11.9% of the glycoproteins released by a monolayer culture of the established breast carcinoma cell line, MCF-7. alpha 1-Acid glycoprotein made up 0.7 to 3.1% of the labeled glycoproteins. alpha 1-Antichymotrypsin is a known neutral serine proteinase inhibitor with a particularly strong affinity for cathepsin G. alpha 1-Acid glycoprotein may function primarily as a potent immunomodulator by suppressing lymphoblastogenesis. These glycoproteins may thus have regulatory roles in the proteolytic modification of breast tissue and represent the tissue's own protecting shield against invading leukocytes.

The efficient isolation of trypsin and chymotrypsin from a crude pancreatic extract was achieved by affinity chromatography on specific adsorbents prepared by coupling of both naturally occurring protease inhibitors and also synthetic low-molecular-weight protease inhibitors to hydroxyalkyl methacrylate gels. Specific sorbents prepared with synthetic inhibitors are stable and are suitable for the isolation of chymotrypsin and trypsin even on a large scale.

A tissue culture line of a human malignant melanoma, SEKI, induced cachexia in nude mice (BALB/c-nu/nu) (Kondo et al., Cancer Res., 41: 2912-2916, 1981). During the investigation of the cause of the cachexia, the melanoma was found to produce a protein immunologically identical to human alpha 1-antichymotrypsin (alpha 1-Ach). Tissues of the SEKI melanoma contained the protein immunologically equivalent to 0.29 +/- 0.11 (S.D.) mg of human alpha 1-Ach per g of wet tissue, while the other six human malignant tumors transplanted into nude mice did not contain a detectable amount of it. In the serum of nude mice bearing the melanoma, this protein appeared soon after the tumor growth occurred and gradually increased up to the level equivalent to 5 mg of human alpha 1-Ach per dl. Removal of the tumor resulted in a rapid decrease of the protein in the serum to an undetectable level within 1 day. This problem was never detected in the serum of nude mice bearing the other 27 human malignant tumors or controls. Purification of this protein was carried out by the column chromatography using DE-52, Blue-Sepharose, and SP-Sephadex. The elution patterns were the same as those of alpha 1-Ach in human serum, and the molecular weight of the protein was estimated as 69,000 by Sephadex G-100 column chromatography and 65,000 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. This purified protein, however, did not exhibit inhibitory activity against chymotrypsin. These results show that this melanoma produced a protein immunologically identical and physicochemically very similar to human alpha 1-Ach. This melanoma-nude mouse system may provide a useful model for investigating the synthesis of human alpha 1-Ach and analysis of its physiological roles.

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.

Via attachment of p-(omega-aminoethyl)phenylboronic acid to CH-Sepharose in the presence of water-soluble carbodiimide, a new sorbent for the biospecific chromatography of serine proteinases was obtained. The sorbent was shown to be suitable for the purification of subtilisn, alpha-chymotrypsin and trupsin. It is assumed that the serine hydroxyl group at the active site of the enzyme forms, with the boronic acid moiety of the ligand, a structure that imitates transition enzyme--substrage complex. The presence of glycerol selectively improves the binding of serine proteinases, presumably because of stabilization of the tetrahedral state of the boron atom. Direct isolation of subtilisin from a Bacillus subtilis cultural filtrate on phenylboronic acid-containing sorbent gives a virtually homogenous enzyme (42-fold purification) in a nearly-quantitative yield.

Recent data on the nature of trypsin-, chymotrypsin-like proteinases of fish are generalized. Localization and secretion of these enzymes in pyloric appendages of fish are considered in detail. Trypsin and chymotrypsin are in the state of proenzymes and transform into the active form by means of their own proteolytic factors. It is observed that the classical methods for isolation of individual chymotrypsin and trypsin cannot be used in the case of fish, since the fish enzymes are stable in the neutral and low-alkaline media and unstable in the acid medium. This is, first of all, accounted for by differences in the physicochemical characteristics of the test enzymes. New data on the biospecific chromatography of serine proteinases of lower invertebrates are presented. Biospecific sorbents used for isolating enzymes from mammals are not always convenient for purification of fish serine proteinases. This evidences for considerable differences in their active sites and, probably, in their binding sites, whose nature is responsible for the specificity and is important for the selective chromatography of enzymes.

The separation of model protein pairs (hemoglobin/albumin, trypsin/chymotrypsin, hemoglobin A/hemoglobin F) was studied in an apparatus for preparative gel electrophoresis at loads up to 40mg/cm2 of the cross-sectional area of the gel bed. Separation was favored by higher ionic strength and by longer migration path. Under the conditions used and within the load range studied, increasing total protein load had no adverse effect but increased voltage gradient, temperature, or gel strength were all unfavorable.

Four active forms of chymotrypsin C (C1, C2A, C2B, and C3) were isolated from the autolyzed porcine pancreas glands. Their molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 29 100 for C1, 26 300 for C2A and C3, and 25 500 for C2B. The kinetic analyses of esterase activity of the enzymes toward Ac-LLeu-OEt and Ac-LPhe-OEt showed that chymotrypsin C1 hydrolyzed the two substrates more efficiently than did chymotrypsin C3. Chymotrypsin C1 consisted of chain A (H-Cys-...-Asn-OH, Mr 886) and chain BC (H-Val-...-Lys-OH, Mr 28 200). Chymotrypsin C3 consisted of the two components of C3L and C3S that could be dissociated in the presence of 2.3% SDS. C3L consisted of the chain A and the chain C (H-Ser-...-Lys-OH, Mr 13 600). C3S was the chain B (H-Val-...-Lys-OH, Mr 11 800). These kinetic and chemical analyses show that chymotrypsins C1 and C3 correspond to chymotrypsin A delta and A alpha, respectively.