Comparative action of cathepsins D, B, H, L and of a new lysosomal cysteine proteinase on rabbit myofibrils.

Specific action of cathespins D, B, H, L, and of a new high Mr (molecular weight relative to hydrogen) cysteine proteinase, on rabbit muscle myofibrils was studied at pH 5·7 by following changes affecting their ATPase activities, their calcium sensitivity, their effect on the ultrastructure, as well as the electrophoretic pattern of the contractile proteins in the presence of SDS. With regard to the MgCa-enhanced ATPase activity, all these proteinases had a very similar effect. A decrease in this activity was thus noted concomitantly with a shift of the straight-line graph obtained when plotting the present acto-myosin ATPase activity versus KCl concentrations. Cathepsins D, B, L and the high Mr cysteine proteinase induced a decrease in both the calcium ATPase activity of myosin and the calcium sensitivity of myofibrils. On the contrary, the Mg-EGTA-dependent ATpase activity was increased. Except for cathepsin H, extensive hydrolysis of proteins occurred in myofibrils treated with each of the lysosomal proteinases tested. However, different specificities could be distinguished. On the one hand, cathepsins D and B affected mainly myofibrillar protein running above and below actin, respectively, on SDS-polyacrylamide gel electrophoresis; on the other hand, the high Mr cysteine proteinase exhibited broader specificity since most of the proteins were hydrolyzed irrespective of their Mr. Myofibrils incubated with cathepsins B and the high Mr cysteine proteinase showed ultrastructural modifications at the level of Z-line, M-bands and A-bands. Myofibrils treated with cathepsin D and cathepsin H appeared almost unaltered. On the basis of these characteristics, cathepsin H hardly affected myofibrils. These results provide evidence for the involvement of the lysosomal proteinases in the meat ageing process and are discussed in regard to the changes occurring at the myofibrillar level during conversion of muscle into meat.

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.

Measurement of autolysis in beef muscle homogenates.

Homogenates of beef longissimus dorsi muscle have been used to study the effect of pH, temperature and Ca(2+) ions on post-mortem autolysis. Measurements of myofibrillar proteins were made after SDS-polyacrylamide gel electrophoresis and it was found that the intensity of the troponin T band could be used as an indicator of autolysis. At pH values below 6·0, the addition of EDTA increased the rate of loss of troponin T: above pH 6·0, the rate of loss of troponin T was accelerated by Ca(2+) ions. It was concluded that there were at least two proteolytic enzyme systems involved-cathepsin B at low pH and a calcium-activated factor at high pH.

Relationship between toughness and troponin T in conditioned beef.

During conditioning of beef, the decrease in toughness paralleled the loss of troponin T with time. The rates of increase with temperature gave an energy of activation of 72 kJ/mol for troponin T breakdown and 63 kJ/mol for the reduction in toughness. The extents of reduction of the two components were significantly related; loss of troponin T accounted for about 60% of the variation in toughness. We concluded that measurement of the loss of troponin T rovides a good indicator of the rate and extent of toughness changes during conditioning.

The influence of pH and temperature on the properties of myosin.

1. The rate of denaturation of myosin solutions at temperatures between 32 degrees and 45 degrees and at pH values between 5.3 and 6.2 has been studied, by using adenosine-triphosphatase activity and solubility in m-potassium chloride at pH6.1 as criteria. 2. Myosin, when heated, loses its adenosine-triphosphatase activity before it becomes insoluble. 3. The loss of adenosine-triphosphatase activity and solubility are both first-order and pH-dependent reactions. Myosin, however, becomes insoluble only when heated within a narrow range of pH values. 4. The thermodynamic functions found for the two processes of denaturation are compared and discussed. 5. The possibility is discussed that, in muscle undergoing rigor, conditions may obtain that would denature myosin.