Purification and characterization of cationic chymotrypsin from the pancreas of the Arabian camel (Camelus dromedarius)

This study reports on the purification and characterization of a cationic enzyme with chymotryptic activity from camel pancreas. The enzyme was purified 52-fold in a 48% yield by a three-step chromatographic procedure consisting of anion-exchange, cation-exchange and affinity chromatographies. The purified enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 7.3 and its molecular mass was found to be 23,600 Da. The enzyme was identified as a cationic chymotrypsin according to its physiochemical properties, substrate specificity and susceptibility to inhibition. It was active towards esters of aromatic amino acids but much less active towards a leucine ester. In all cases, the k_cat values of the camel enzyme were less than the corresponding values of bovine chymotrypsin A. It also showed a lower level of kininase activity. Camel chymotrypsin was more susceptible than its bovine equivalent to inhibition by soybean trypsin inhibitor and aprotinin. It showed the same pH optimium as bovine chymotrypsin A for its esterolytic activity, but was more dependent on CaCl₂ for long-term stability.     

Purification and characterization of Locusta migratoria chymotrypsin

A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.     

Purification and properties of protease F, a bacterial enzyme with chymotrypsin and elastase specificities

It has been previously demonstrated that commercial bacterial fibrinolysin (EC 3.4.21.7) selectively cleaves the bond between Met-53 and Ala-54 in ovine prolactin (199 amino acids). A one-step purification procedure on DEAE-cellulose for Protease F, which is the active component of bacterial fibrinolysin, and properties of the purified enzyme are reported. The enzyme is homogeneous as judged by acrylamide gel electrophoresis. Its molecular weight, calculated from gel filtration experiments on Sephadex G-100, is around 13,800. Amino acid analyses do not reveal the presence of any half-cystines. The presence of one tryptophan residue per enzyme molecule was resolved from the fluorescence spectrum. Amino terminal analysis showed that leucine was at the amino terminal position. Protease F hydrolyzes casein and synthetic specific substrates for chymotrypsin and elastase esterases but not for trypsin esterases. It is fully inhibited by phenylmethylsulfonyl fluoride, by chicken ovoinhibitor, and by Chymotrypsin Inhibitor I from potatoes but not by the trypsin-chymotrypsin inhibitors from soybeans and chick peas or by tosyl-L-phenylalanine chloromethyl ketone. The enzyme is stable at room temperature and in the cold, it is not affected by dialysis or by freezing and thawing, but it is inactivated during freeze-drying. The circular dichroism spectra of Protease F indicate an approximate 20% alpha-helix content of the enzyme with a considerable similarity to those of subtilisin, elastase, and beta-trypsin. The relatively low molecular weight of Protease F, the absence of intrachain disulfide bridges, and the fact that it is inhibited by several, but not all, chymotrypsin inhibitors suggest that it may differ phylogenetically from the known serine proteases.     

Some kinetic properties of dogfish chymotrypsin

A comparison of some kinetic properties was made between bovine chymotrypsin and chymotrypsin isolated from the spiny dogfish (Squalus acanthias). The major difference between the two enzymes was observed in the molecular activity (kcat), with the dogfish enzyme being two to three times more active than the bovine enzyme. The exact difference was dependent on the substrate and assay conditions. The two enzymes showed similar kinetic properties with respect to the following: similar inhibition by indole and naphthol derivatives, activities vs BTEE and a series of n-fatty acid esters of p-nitrophenol, KM values, optimal pH and temperature and activation energies.     

Purification and characterization of chymotrypsin inhibitors from marine turtle egg white

The egg white of marine turtle(Caretta caretta Linn.) contains two chymotrypsin inhibitors and one trypsin inhibitor. The two chymotrypsin inhibitors were purified to homogeneity, as judged by ion-exchange chromatography, Polyacrylamide gel and sodium dodecyl sulphate-gel electrophoresis, isoelectric focusing, immunochemical tests and sedimenttation in the ultracentrifuge. Their sedimentation coefficient values were independent of protein concentration. Their amino acid composition was similar, and contained seven disulphide bonds, and methionine and carbohydrate moiety were absent. Each inhibitor consisted of a single polypeptide chain of 117 amino acids. The average molecular weight of each inhibitor, calculated from sedimentation and diffusion coefficient values, amino acid composition and sodium dodecyl sulphate-gel electrophoresis was 13000. Both the inhibitors were stable over the pH range of 2–11. They inhibited α-chymotrypsin by forming enzymeinhibitor complexes at a molar ratio of unity. The dissociation constant of each complex was 1.06 × 10⁻¹⁰ M. Both the inhibitors were indistinguishable in their physical, chemical and inhibitory properties except for their isoelectric points which were pH 5.23 for inhibitorA and pH 6.0 for inhibitorB. Chemical modification of all amino groups with trinitrobenzene sulphonate had no effect on their inhibitory activity     

Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.     

The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Purification and properties of the inhibitors.

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50% of the isolated inhibitors and were further purified to a homogeneous state. The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 degrees C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HCl. The intact inhibitors were destroyed when the peas were cooked at 100 degrees C or when they were toasted at 130 degrees C. The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20% of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).     

Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer [corrected

An elastase-like chymotrypsin was purified by aprotinin-agarose affinity chromatography from the midgut extract of cardamom shoot and capsule borer, Conogethes punctiferalis. The purified enzyme had a Vmax of 687.6 +/- 22.1 nmole pNA released/min/mg protein, Km of 0.168 +/- 0.012 mM with SAAPLpNA as substrate and gave a single band on SDS-PAGE with a molecular mass of 72.1 kDa. Casein zymogram revealed one clear zone of proteolytic activity, which corresponded to the band obtained with SDS-PAGE indicating that this could be a single-polypeptide enzyme.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

Purification of chymotrypsin by subunit exchange chromatography on the denatured protein

The alpha-chymotrypsin subunits immobilized under denaturing conditions (6 M urea or 1% SDS) on CNBr-activated Sepharose 4B, were found to interact with soluble chymotrypsin subunits with the formation of oligomers higher than dimers. Subunits immobilized under nondenaturing conditions form only dimers. The effects of several parameters, such as organic solvents, cations, and anions of the lyotropic series, on the associating properties of the immobilized derivatives were examined. The interaction between immobilized and free enzyme was shown to be specific because extraneous proteins and compounds were not bound by the derivatives. Chymotrypsinogen, studied analogously, did not show appreciable self-associating capacity. Chymotrypsin subunits immobilized under denaturing conditions and packed in a column proved to be suitable for the purification of chymotrypsin from both bovine and porcine pancreatic extracts. The "subunit exchange" chromatography of such extracts, carried out between pH 2.5 and 4, gave an eightfold purification with a 93% recovery of chymotryptic activity. The specific activity was ca. 12,000 Schwert and Takenaka units/mg. Only 6% of the tryptic activity was bound by the column. The capacity of the matrix, 6 mg chymotrypsin/mL, dropped to about 70% of the original value after.     

Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.     

Purification and pH stability characterization of a chymotrypsin inhibitor from Schizolobium parahyba seeds

Schizolobium parahyba chymotrypsin inhibitor (SPCI) was completely purified as a single polypeptide chain with two disulfide bonds, by TCA precipitation and ion exchange chromatography. This purification method is faster and more efficient than that previously reported: SPCI is stable from pH 2 to 12 at 25 degrees C, and is highly specific for chymotrypsin at pH 7-12. It weakly inhibits elastase and has no significant inhibitory effect against trypsin and alpha-amylase. SPCI is a thermostable protein and resists thermolysin digestion up to 70 degrees C.     

Purification and sequence of an opioid peptide derived from ovine proenkephalin

An enkephalin-containing peptide originating from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: GLY-GLY-GLU-VAL-LEU-GLY-LYS-ARG-TYR-GLY-GLY-PHE-MET (preproenkephalin 128-140) which represents a portion of peptide F (preproenkephalin 107-140). This peptide has a sequence identical to that of bovine preproenkephalin 128-140 while it differs from the corresponding human sequence in positions 129, 131 and 133.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

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.     

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.