David J. Etherington

Examination of cathepsins B, D, H and L activities in dry-cured hams.

Cathepsins B, D, H and L as well as the glycosidases β-glucuronidase and N-acetyl-β-glucosaminidase were assayed for activity in fresh pork muscles stored for up to 20 days at 4°C and in 3- and 8-month dry-cured hams. Cathepsin B, H and L activities fell by 40-79% after 20 days while cathepsin D activity remained unchanged. All the enzymes were still active after 8 months of dry curing; with the recoveries found to be in the range 14-73%. Contaminant microorganisms did not appear to contribute to the observed proteinase activities. Cathepsins B, H and L activities from the 8-month ham have been confirmed as cysteine proteinases by the use of specific inhibitors. It was further confirmed that the cathepsin B and L activities in fresh pork and dry-cured ham were similar by isoelectrofocusing. We suggest that the presence of the curing salts may be important in stabilising the muscle enzymes during the curing process.

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non‐helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37°C and pH values from 3.5 to 7.0. Cleavage occurs only within the non‐helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Conditioning of meat from different species. Relationship between tenderising and the levels of cathepsin B, cathepsin L, calpain I, calpain II and β-glucuronidase

The conditioning times for beef, calf, lamb, pig and chicken M. pectoralis profundus were determined from extensibility measurements at 15°C. Extracts from the same muscle from these animals and also rabbit were assayed for free and total cathepsin activities using a new method to determine the extent of inhibition by cystatins. A new method was also developed for the rapid estimation of each calpain. The results show that pig muscle contained most cathepsin activity. Cathepsin L appeared to be inhibited to a greater degree than cathepsin B in the pig, rabbit and chicken muscle extracts. These extracts also contained proportionately more cathepsin L than the other species. Chicken muscle contained only a low level of the more sensitive neutral proteinase, calpain I. It was concluded that the rapid conditioning time for chicken muscle could not be explained in terms of either the free or total levels of cathepsins B and L, nor of the individual calpain activities.

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates.

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Proteinase (Cathepsin B, D, L and Calpains) levels and conditioning rates in normal, electrically stimulated and high-ultimate-pH chicken muscle

Control, electrically stimulated (ES) and glycogen-depleted (GD) chicken muscles were conditioned at 15°C with continuous mechanical testing for extensibility. The ES and GD muscles went into rigor 3·6 and 2·8 h earlier, respectively, than control muscle. At 24h post-rigor the extensibility of control muscle (11·2%) was markedly less than ES (19·2%) and GD (27·3%) muscles indicating that these latter two treatments should provide more tender meat. Measurement of sarcomere lengths showed no significant differences between control and GD muscle and thus, the greater extensibility in the high pH condition may be restricted to a wider separation of myofibriller fragments at the intermittent fracture zones when under load. Examination of muscle proteinase (cathepsins B, D and L, calpains I and II) and glycosidase (β-d-glucuronidase, N-acetyl-β-d-glucosaminidase) levels at 0 and 48h post-slaughter revealed changes in some key enzymes between the different treatments. Calpain I activity declined markedly during 48h storage of ES muscle (83%) compared to control (58%) and GD (63%) muscles. Cathepsin B and L activities did not decline during storage of ES muscle but there was a slight fall in control and GD muscles. Dosing of chicken shortly before slaughter with inhibitors of cysteine proteinases had a negligible effect on conditioning rate, apparently due to lack of inhibition of these proteinases during this short time period in the intact muscle.

Collagenolytic cathepsins of rabbit spleen: a kinetic analysis of collagen degradation and inhibition by chicken cystatin

We have investigated the steady state kinetics of the degradation of native fibrillar collagen at pH 3.4 by four collagenolytic cathepsins of rabbit spleen. For each enzyme, the dependence of initial velocity on collagen concentration was well described by the Michaelis-Menten mechanism. Km, expressed as the concentration of triple-helical chains, and kcat values were determined for cathepsins B, L, N and S. The ratio of Kcat to Km suggest that cathepsins L and N are far more effective at collagen solubilization than either cathepsins S or B. Ki values were determined for the inhibition of collagenolytic activity at pH 3.4 using cystatin, a naturally-occurring cysteine proteinase inhibitor. All four cysteine proteinases were inhibited by cystatin in this assay system, although it was found to be a tighter binding inhibitor of cathepsin L, than for cathepsins N and S (approximately 5-fold less), or cathepsin B (approximately 500-fold less).

The solubilization of myofibrillar proteins by calcium ions.

The effect of elevated levels (30 mm) of Ca(2+) and other divalent metals ions on rabbit psoas myofibrils was studied to determine whether these caused solubilization of structural proteins and if so whether the effect was due to salting-in or to proteolytic fragmentation resulting from activation of calpains. Incubation of myofibrils in 30 mm CaCl(2) at either pH 5·6 or 7·0 did not cause any apparent solubilization of the major Z-disc proteins, but there was an immediate ( < 1 min) solubilization of C-protein and troponin I together with small amounts of Mr 80 000 protein, troponin T and tropomyosin. Longer incubations with CaCl(2) extracted little additional C-protein but there was a steady increase with time in the solubilization of proteins with Mr values of 45 000 and 42 000, troponin T, tropomyosin and troponin I. Another high molecular weight protein of Mr 3-400 000 was extracted at pH 7·0 but not at pH 5·6. Similar results were obtained on incubation with 30 mm MgCl(2). In contrast to these findings, the same concentration of ZnCl(2) caused no detectable solubilization of myofibrillar proteins. The inclusion of proteinase inhibitors, E64, leupeptin, pepstatin or PMSF did not prevent the immediate solubilization of proteins. This showed that the solubilization of the proteins by Ca(2+) ions was due to salting-in rather than to proteolytic action by calpains.

Purification and immunological characterisation of two calcium-activated neutral proteinases from rabbit skeletal muscle.

Two Ca2+-activated neutral proteinases have been prepared to a high degree of purity from rabbit skeletal muscle. One, calpain I, is optimally activated by 100 microM Ca2+ and the other, calpain II, by 1 to 2 mM Ca2+. Both enzymes have two subunits of molecular weight 80 000 and 28 000. Antibodies have been raised against the native forms of both enzyme. It was found that the antibody to native calpain I reacted only with calpain I and not with calpain II, and similarly the antibody to native calpain II reacted only to calpain II. This suggested that the epitopes in the two enzymes are located in regions that are structurally different. However, immunoblotting of the denatured calpains after SDS-polyacrylamide-gel electrophoresis revealed cross-reaction between the two subunits for both enzymes. Therefore, although the denatured enzymes have common antigenic sites it would appear that these are not exposed equally in the native proteins.

Purification and characterisation of collagenolytic cathepsins from rabbit liver

Following the early characterisation of cathepsln B as a cysteine proteinase, at least 5 more enzymes in this class have been described, designated cathepsins H,L,N,S and T [1-4]. We have shown that the collagenolytic activity of bovine spleen was caused by cathepsin B and cathepsin N [5]. In [6], cathepsin L isolated from rat liver was also reported to degrade collagen. We have now investigated the cysteine proteinases of rabbit liver, with particular emphasis on the major sources of collagenolytic activity in this tissue.

Production of a monospecific antiserum to cathepsin L: The histochemical location of enzyme in rabbit fibroblasts

A polyclonal antibody against rabbit cathepsin L was raised in goats and shown to be specific for both active and inactive enzyme. Using this antibody we have examined the distribution of cathepsin L in primary rabbit skin fibroblasts by immunohistochemistry and found that all the enzyme is located within lysosomal granules. At confluence many cathepsin-L-containing lysosomes were seen in each celt. A new but nonspecific histochemical substrate for cathepsin L was tested and a similar distribution was obtained. Our results indicate that the immunohistochemical technique can be reliably employed for the specific location of cathepsin L in cells and tissues.