Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels

The structure of muscle injected with specific cysteine protease inhibitors was examined to determine whether inhibitors cause denaturation and the de gradation post-mortern of myofibrillar proteins was followed using SIDS ele ctrophoresis. Given the central role of calcium in theories of tenderisatio n the level of free calcium was measured during the early post-mortem perio d. The protease enzyme inhibitor E-64 was injected into the m. longissimus et thoracis lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe -Ala-CHN2. The left LTL (control) was injected with saline (0.25 M NaCl). M uscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 da ys post-mortem (n=215). Muscle samples were selected from eight portions of the LTL (1-day post-mortem, from six different carcasses) for examination by transmission electron microscopy. Matching light images of myofibrils we re obtained after determination of myofibrillar fragmentation. Free calcium concentration was determined for all samples (n=191) using an ion selectiv e electrode excluding those 'at death'. Light images of myofibrils from tre ated samples showed normal striations and no evidence of denaturation or ag gregation compared to control samples. This also applied to the samples pro cessed for examination by electron microscopy. Appearance of the 30-kDa sub unit increased with time (P <0.001) post-mortem. The interaction between ag eing and stimulation had an effect (P <0.00 1) on the amount of a protein d esignated M 1. The amount of M I measured pre-rigor was greater for stimula ted muscle, but the rate of decline was also greater through to day 2 post- mortem, Proteolysis was very rapid in the first 24 h post-mortem in ovine m uscle. Ageing had an effect (P <0.001) on the free calcium concentration, w hich increased as muscle aged. As a covariate pH also had an effect (P <0.0 5). Based on a non-linear model when the concentration of free calcium reac hed a plateau (similar to 110 muM) the predicted pH was 5.5 (ultimate). Fro m the qualitative observation of images and the levels of free calcium in i njected muscle there is no support for the view that the inhibitors bind to sarcomere proteins, occupying sites to which calcium might bind. The level s of free calcium do not provide support for the view that m-calpain has a role in post-mortem. tenderisation, but do suggest along with results of pr otein degradation that activation of mu -calpain is likely to occur before the pH drops to 6.26.1. (C) 2001 Elsevier Science Ltd. All rights reserved.