On the specificity of bovine spleen cathepsin B2.

The specificity of bovine spleen cathepsin B2 has been investigated by means of some natural oligo- and polypeptides, i.e. glucagon, melittin, insulin A and B chain, bradykinin, angiotensin I and II, oxytocin ACTH, clupein and salmin. The enzyme is primarily a carboxypeptidase which hydrolyzes peptide linkages of most amino acids common to proteins. In addition, cathepsin B2 displays amidase and esterase activity without requiring a free carboxyl group. The main pH optimum is between 4 and 5, in some cases higher.     

Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.

Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

Structure-activity relationship in buffalo spleen cathepsin B.

Effect of pH, urea, and guanidine hydrochloride on the activity and structure of buffalo spleen cathepsin B was investigated. At alkaline pH, there was an irreversible loss of the structure as well as the activity of the buffalo enzyme. At acidic pH, however, the inactivation of the enzyme was reversible. The enzyme reversibly lost most of its activity at denaturant concentrations which did not cause a significant change in its secondary structure. The inactivation could be attributed to minor perturbations in the environment of the amino acid residue(s) at and/or around the active site of the enzyme. High urea/guanidine hydrochloride concentrations leading to the structural changes in cathepsin B made the inactivation process irreversible.     

Structures at the proteolytic processing region of cathepsin D

The amino acid sequences at the "proteolytic processing regions" of cathepsin Ds have been determined for the enzymes from cows, pigs, and rats in order to deduce the sites of cleavage as well as the function of the proteolytic processing of cathepsin D. For bovine cathepsin D, the "processing region" sequence was determined from a peptide isolated from the single-chain enzyme. The COOH-terminal sequence of the light chain and the NH2-terminal sequence of the heavy chain were also determined. The processing region sequence of porcine cathepsin D was determined from its cDNA structure, and the same structure from rat cathepsin D was determined from the peptide sequence of the single-chain rat enzyme. From sequence homology to other aspartic proteases whose x-ray crystallographic structures are known, such as pepsinogen and penicillopepsin, it is clear that the processing regions are insertions to form an extended beta-hairpin loop between residues 91 and 92 (porcine pepsin numbers). However, the sizes of the processing regions of cathepsin Ds from different species are considerably different. For the enzymes from rats, cows, pigs, and human, the sizes of the processing regions are 6, 9, 9, and 11 amino acid residues, respectively. The amino acid sequences within the processing regions are considerably different. In addition, the proteolytic processing sites were found to be completely different in the bovine and porcine cathepsin Ds. While in the porcine enzyme, an Asn-Ser bond and a Gly-Val bond are cleaved to release 5 residues as a consequence of the processing; in the bovine enzyme, two Ser-Ser bonds are cleaved to release 2 serine residues. These findings would argue that the in vivo proteolytic processing of the cathepsin D single chain is probably not carried out by a specific "processing protease." Model building of the cathepsin D processing region conformation was conducted utilizing the homology between procathepsin D and porcine pepsinogen. The beta-hairpin structure of the processing region was found to (i) interact with the activation peptide of the procathepsin D in a beta-structure and (ii) place the Cys residue in the processing region within disulfide linkage distance to Cys-27 of cathepsin D light chain. These observations support the view that the processing region of cathepsin D may function to stabilize the conformation of procathepsin D and may play a role in its activation.