“…The other cleavages indicated sequential removal of single amino acid residues from the N-terminus, characteristic of an aminopeptidase. However, it should be noted that an internal cleavage site between Glu13-Ala14 has been observed for np cat H, indicating that the native enzyme is also an endopeptidase, in agreement with previous observations ( , ). 6 Cleavage of the oxidized insulin β chain by recombinant human (rh cat H) and native porcine (np cat H) cathepsins H. The cleavages determined after 30 min of incubation are shown.…”
Section: Resultssupporting
confidence: 92%
“…However, the same cleavage (Glu13-Ala14) has also been observed with purified porcine enzyme, although to a minor extent. This is in agreement with previous reports on the endopeptidase activity of native cathepsin H (31,32,48). From the crystal structure of the complex between cathepsin H and stefin A, it is evident that the mini chain of cathepsin H is flexible and can be moved from its normal position (45) to accommodate the N-terminus of the inhibitor.…”
Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.
“…The other cleavages indicated sequential removal of single amino acid residues from the N-terminus, characteristic of an aminopeptidase. However, it should be noted that an internal cleavage site between Glu13-Ala14 has been observed for np cat H, indicating that the native enzyme is also an endopeptidase, in agreement with previous observations ( , ). 6 Cleavage of the oxidized insulin β chain by recombinant human (rh cat H) and native porcine (np cat H) cathepsins H. The cleavages determined after 30 min of incubation are shown.…”
Section: Resultssupporting
confidence: 92%
“…However, the same cleavage (Glu13-Ala14) has also been observed with purified porcine enzyme, although to a minor extent. This is in agreement with previous reports on the endopeptidase activity of native cathepsin H (31,32,48). From the crystal structure of the complex between cathepsin H and stefin A, it is evident that the mini chain of cathepsin H is flexible and can be moved from its normal position (45) to accommodate the N-terminus of the inhibitor.…”
Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.
“…This led us to include in our purification procedure the use of an affinity column made of anti‐BSA IgG covalently linked to activated sepharose 4B. Serum obtained from rabbits immunised with cathepsin L purified in this way was tested against both a bovine kidney crude extract and different dilutions of purified cathepsin L. As can be observed in Fig 6, only one main precipitating arch was observed this time for a bovine kidney crude extract, which was coincident with the precipitation arch corresponding to pure cathepsin L. This proved the quality and high specificity of the serum for the recognition of native cathepsin L from bovine species, and the proof that closely related peptidases such as cathepsin B, H, S or V do not cross‐react with anti‐cathepsin L antibodies, as already pointed out previously by other authors 3, 40, 49, 50…”
Section: Resultssupporting
confidence: 85%
“…This protein is extremely immunogenic,48 and its presence in trace amounts in our final cathepsin L preparation would have been enough to generate an appreciable immune response. Similar to us, Lokshina et al 3 found that antiserum developed against highly purified cathepsin L from bovine spleen also reacted with another bovine protein of higher M r than cathepsin L, but they did not clarify the identity of this antigen. By means of radial immunodiffusion we effectively determined a concentration in our final cathepsin L preparation of 8 µg BSA ml −1 purified protein.…”
Section: Resultsmentioning
confidence: 69%
“…Most of them have a cysteine residue responsible for the catalysis and belong to the cysteine peptidase family C1 or ‘papain family’ with similar amino‐acid sequences and folds 1. In spite of this, differentiation between them is possible through the development of specific antibodies 2, 3. They catalyse the hydrolysis of peptide, amide, ester and thiol ester bonds and are responsible, together with other lysosomal proteolytic enzymes, for the degradation of proteins as far as the generation of free amino acids and dipeptides, which are small enough to diffuse through the lysosomal membrane and to be used again for protein synthesis.…”
Two approaches have been carried out for the production of polyclonal antibodies specific of bovine cathepsin L (EC 3.4.22.15). The first one consisted in the purification of cathepsin L from bovine liver, through the development of an original protocol that can be achieved in 4 days, and further injection in rabbits. The purification scheme included the preparation of an enriched lysosomal extract, selective precipitation with ammonium sulfate and different chromatographic steps including size-exclusion chromatography, cation-and anion-exchange chromatography and the use of an affinity column for the removal of trace amounts of bovine serum albumin. The final enzyme preparation was purified 8400-fold and consisted predominantly in the double-chain form of cathepsin L. This was proved by the presence of a main protein band with M r of 25 kDa, corresponding to the cathepsin L heavy chain, in SDS-PAGE under reducing and denaturant conditions, and was further confirmed by immunoblotting. The second approach consisted in the generation of polyclonal antibodies against the peptide sequence 99 PECSAANDTGFVDIPQR 115 , specific of the bovine cathepsin L heavy chain. Both types of antibodies were proved to specifically recognize cathepsin L. However, while antibodies raised against the whole protein recognized native cathepsin L, the antipeptide was able to recognize the enzyme in its denatured form. Both of them are adequate for the future development of immunoassays, allowing a rapid and accurate quantification of cathepsin L levels directly in crude extracts of meat and meat-derived products.
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