Porcine Chymotrypsin Research Articles

Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.

A functional comparison of ovine and porcine chymotrypsins

Chymotrypsin was isolated from ovine and porcine pancreas by affinity chromatography on immobilised 4‐phenylbutylamine. The apparent molecular weights of the two proteins were estimated by SDS PAGE to be 25.7 and 27.3 kD respectively. Specific activities for ovine and porcine chymotrypsins (OC and PC) were 32.8 and 31.2 μmol/min per mg, respectively, at pH 8.5 and 25°C using 0.1 mM N‐succinyl‐Ala‐Ala‐Pro‐Phe‐pNA (SAAPFpNA) as substrate. Values of the Michaelis constants for ovine and porcine chymotrypsins with SAAPFpNA were comparable at pH 8.0 and at temperatures between 20 and 45°C, as were the kcat values. Both enzymes were stable under acidic conditions but were susceptible to thermal denaturation above 45°C. Hydrolysis of lysozyme and casein by ovine or porcine chymotrypsin yielded very similar fragmentation profiles as determined by RP‐HPLC.

Purification and partial characterization of chickpea 2S albumin.

A chickpea 2S albumin has been purified by solubilization in 60% methanol and ion-exchange chromatography. Under denaturing conditions it is composed of two peptides of 10 and 12 kDa. Native molecular mass determined by gel filtration chromatography is 20 kDa. Amino acid composition shows that it is rich in sulfur amino acids, mainly cysteine with 4.6% of the total. On the other hand, it has antinutritional characteristics of being allergenic for chickpea-sensitive individuals and inhibitory against porcine chymotrypsin with a lesser degree toward trypsin. The results of interest from a nutritional point of view are discussed.

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.

Serine protease inhibitors of North American leeches

1. 1. Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. 2. 2. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. 3. 3. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. 4. 4. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.

The substrate specificity of proteinase B from Baker's yeast

The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The K m value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1 20 that of chymotrypsin A. K m and k cat for Bz-Arg-OEt are 1 50 and 1 7 as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.

The preparation of trypsins and chymotrypsins from bovine and porcine residues after insulin extraction.

Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymotrypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP) - Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI) - Sepharose. The bovine proteinase powder contained alpha-chymotrypsin, trypsin and chymotrypsin B in the ratio 5 : 2 : 1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1.4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-1 and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine alpha-chymotrypsin, a three chain structure, rather than porcine chymotrypsin Api, a two chain structure. Futhermore, the B-chain appears to be cleaved, possibly at residues Phe89-Lys90.

Porcine chymotrypsin A-pi, a more acidic chymotrypsin.

A kinetic study of procine chymotrypsin A-pi revealed two characteristic properties of this type of chymotrypsin: 1. Porcine chymotrypsin A-pi, like bovine chymotrypsin B-pi does not bind proflavin, which is a competitive inhibitor of bovine trypsin and chymotrypsin A-alpha. 2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.

Preparation and properties of three-chain neochymotrypsinogens

Porcine chymotrypsin C is able to hydrolyze in native bovine chymotrypsinogen A bonds 146 and 148 and also bonds 10 and 13 situated a few residues apart from the “activating” bond Arg 15Ile 16. Hydrolysis of bond 13 does not activate chymotrypsinogen. But it is compatible with subsequent activation of the molecule and, therefore, gives rise to a neochymotrypsinogen with three open chains. Residues Tyr 146 and Arg 145 can be removed from the above neochymotrypsinogen by the combined action of carboxypeptidases A and B, without impairing the activability of the molecule. This observation does not confirm the postulation according to which residue Arg 145 plays a role during chymotrypsinogen activation by trypsin.

L'hydrolyse des liaisons de la leucine par diverses chymotrypsines

The A chains of bovine chymotrypsin A and B and porcine chymotrypsin A (residues 1 to 15) were obtained after performic acid oxidation of their N-terminal half-cystine, and used as substrates for studies with these three chymotrypsins. Two bonds involving the carboxyl group of leucine (bond 13 and bond 10) were cleaved. Bovine chymotrypsin A hydrolyzed bond 13 preferentially; bovine chymotrypsin B hydrolyzed bond 10 preferentially and porcine chymotrypsin A split both bonds at equal rates though very slowly. Thus, in contrast to what is observed for most “aromatic” bonds, the cleavage of leucine bonds by chymotrypsin appears to depend on the structure of the substrate in the vicinity of the bond and on the specificity of the chymotrypsin used. This specificity is different for the three chymotrypsins studied. The remarkable ability of bovine chymotrypsin A to cleave bond 13 in its own chain is related to the high rate at which bovine chymotrypsin A π autolyzes to give chymotrypsin of the δ type. The other chymotrypsins do not autolyze when subjected to the same conditions.

Le chymotrypsinogène a de porc purification et études de quelques propriétés

1.5 g of a weakly cationic chymotrypsinogen (chymotrypsinogen A) and 5 g of trypsinogen can be obtained in essentially homogeneous form from 1 kg of fresh pig pancreas by a single chromatographic step on CM-cellulose. The molecular weight of the porcine chymotrypsinogen A is between 24 000 and 25 000 as determined by ultracentrifugation and by filtration through Sephadex G-100. The amino acid composition resembles those of bovine chymotrypsinogens A and B rather than that of porcine chymotrypsinogen C. Porcine chymotrypsinogen A and bovine chymotrypsinogens A and B contain the same number of histidines (2), tryptophans (8) and disulfide bridges (5), but porcine chymotrypsinogen A contains 5 tyrosines (instead of 4 in bovine A and 3 in bovine B), 2 methionines as in bovine A (compared with 4 in bovine B) and 16 prolines (compared with 9 in bovine A). The N-terminal sequence (first 17 residues) of porcine chymotrypsinogen A and bovine chymotrypsinogens A and B are the same with two exceptions: glutamine at position 7 of bovine chymotrypsinogens A and B is replaced by proline in the porcine chymotrypsinogen and serine at position 14 in porcine chymotrypsinogen A and bovine chymotrypsinogen A is replaced by alanine in bovine chymotrypsinogen B. The arrangement of the 16 residues in the vicinity of the “histidine loop” is identical with that already found by others in bovine chymotrypsinogens A and B. The three chymotrypsinogens studied so far are activated by the cleavage of the Arg 15Ile 16 bond. However, porcine chymotrypsin A π and bovine chymotrypsin B π, in contrast with bovine chymotrypsin A π, do not autolyze and, consequently, do not form a chymotrypsin δ.