Effect of a High-Pressure Treatment on the Intramuscular Proteasome and Its Relationship to Meat Tenderization

Effect of a High-Pressure Treatment on the Intramuscular Proteasome and its Relationship to Meat Tenderization Shuhei Yamamoto Living skeletal becomes a highly nutrious food when converted to meat after slaughter. However, the muscle is instantaneously unsuitable for edibles becaouse of the development of progressive stiffening of muscle, rigor mortis, early in the postmortem period. The postmortem Period that the muscle, required for the softning of rigor moritis is called "aging" or "conditioning". Generally, postmortem tenderization of meat during aging is caused by the proteolytic enzymes. Mainly two enzymes systems, cathepsin and calpain, have been implicated. Catheptic enzymes are released from lysosomes, due to the rupture of lysosomal membrane and may promote aging by proteolysis of myosin, actin, α-actinin, torponin T and troponin I. Calpain removes the Z-disk from myofibrils in the presence of Ca2+ abd causes many changes in myofibrillar structure which could be related to increased meat tenderness. Recently, proteasome or multicatalytic proteinase (MCP) has been added to the candidate for proteolytic system responsible for meat tenderization. Proteasome was first isolated from bovine pituitaries. This ensyme is a high-molecular-mass intracellular proteinase (20S proteasome; 700 kDa), which has a complex subunit compotition and multicatalytic proteolytic activities with different speciticities. The proteasome has two unique enzymological properties as protease: multiple peptidase activities and a latent from. The proteasome has at least five activities - peptidylglutamyl peptide hydorolase, trysin-like, chymotrypsin-like, chymotrypsin-like, branched-chain amino acid-preferring, and small neutral amino acid-preferring activities - within a single proteasome complex. The proteasome isolated from tissues in a latent from can be activated by various chemicals including polylysine, SDS and fatty acids, and treatments such as heat and high hydrostatic pressure. Several reseachers have studied the properties of proteasome prepared from various species or organs.Incidentally, aging is a very important process for meat indusries, especially for beef productions. The availability of intramuscular proteinase for meat aging has been well investigated. Especially, cathepsin and calpain are assumed to participate in proteolysis during meat aging. However, the role of proteasome on meat aging is not yet fully elucidated. In addition, as for bovine skeletal muscle proteasome, nothing has been investigated despite the importance in meat industries. High hydrostatic pressurization is one of the new technologies for tenderizing or accelerating of meat aging which is a very important process for the meat industry. Effects of a high-pressure treatment on cathepsins and calpain which implicate meat tenderization have been well investigated. As for lysosomal enzymes, the pressure-induced increase in proteolytic level in the muscle is due to a release of cathepsins from lysosomes. As for calpain systems, the direct evidence for the pressure-induced Ca2+ release form the sarcoplasmic reticulum has been provided by using electron micrographs of the pyroantimonate-fixed fiber bundles prepared from pressurized muscle tissue. Ca2+ dispersion intomyofibrils may cause an increase in activated calpain. Furthermore, it have been suggested that the calpain level is retained in muscle pressurized up to 200 Mpa, whereas the calpastaitin level was lowered by pressurization. Therefore, the total calpain activity in pressurization muscle appears to be increased by a pressure treatment, and this may result in tenderization of the meat. However, it is not yet clear why the proteasome is activated by high hydrostatic pressurization. This thesis describes 1) the purification and properties of bovine skeletal muscle proteasome in comparison with that of other species or organs, 2) the effects of high hydorstatic pressure treatment on the structure and activities of purified rabbit skeletal muscle proteasome, in comparison with those of heat treatment, 3) the effect of pressure treatment and postmortem aging on activities of muscle proteasome.Capter 1. Purification and Properties of Bovine Skeletal Muscle Proteasome This chapter describes the purification and properties of a multicatalystic proteinase complex, proteasome from bovine skeletal muscle, in comparision wit proteasome prepared from other species or organs. The purified bovine skeletal muscle proteasome exhibited a single band on polyacrylamide gel electrophoresis under nondenaturing conditions. Bovine skeletal muscle proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the activity at pH 5.5 still retained 78～10% of the optimal activity at pH8.0, indicating the possibility that the proteasome is active in muscle during aging. When the proteasome was heated at 60℃ for 15 or 30 min and treated in the presence of 0.0125% SDS, the activity increased over 1.8 and 3.1 times (LLvy as a substrate), respectively. These results on the activation with heat or SDS indecate that the hydrotic activity of proteasome was stimulated under the mild denaturing treatment. The characteristics of the bovine skeletal muscle proteasome obtained in our experiment were almost the same as those of the proteasome prepared from other species or organs.Chapter 2. Effect of a High-pressure Teratment on Activity and Structure of Purified Rabbit Skelatal Muscle Proteasome This chapter was conducted to assess the effect of high hydrostatic pressure on the activity and structure of rabbit skeletal muscle proteasome. Pressure effects on the activity were measure by the fluorometric products released from sythetic substrates under pressure and from fluorescein isothiocyanate (FITC)-labeled casein after release of pressure. Effects on structure were measured by fluorescence spectroscopy under pressure, and by circular dichroism (CD) spectroscopy and surface hydrolyzing after release of pressure. The optimal pressure of synthetic peptides hydrolyzing activity was 50 MPa. The degradation of FITC-labeled casein increased linearly with an increase in pressure applied up to 200 MPa. However, activity markedly decreased at 400 MPa. Concerning the structure, changes in the tertiary structure detected with fluorometric measurement were irreversible, whereas changes in the secondary structure were little compared with that of heat treatment. Therefore the pressure-induced activation of proteasome seemed to be due to a little unfolding of the active sites.Chapter 3. Effect of Pressure Treatment and Postmortem Aging on Activities of Muscle Proteasome This chapter describes the activities of rabbit or bovine skeletal muscle proteasome extracted from pressurized and aged muscle. Furthermore, this chapter discusses whether the high hydrostatic pressure treatment is practically available for meat tenderization or not. Crude proteasome was prepared from pressurized and aged rabbit or bovine skeletal muscle. The results obtained using crude proteasome seems to reflect the intracellular condition of meat than purified proteasome, because crude proteasome activity can be measured without prior purification which may alter the activity. The synthetic peptide hydrolyzing activities of rabbit proteasome still remained in the muscle exposed to pressure up to 100 MPa. However, when a pressure of 400 MPa or more was applied, proteasome was inactivated markedly. The extraction of proteasome from excessive pressurized muscle seems to have been difficult. The proteasome in the aged muscle remained relatively stable throughout the aging process, with the activities after 168 h (7 days) being 35%, 48%, 52% and 30% of the 0 hr postmortem LLVY, LSTR, AAF and LLE hydrolyzing total activities, respectively. The synthetic peptide hydrolyzing activities of bovine muscle proteasome was similar to those of rabbit skeletal muscle proteasome. The obtained results in this experiment suggest that the synthetic peptide hydrolyzing activities still remained in muscle exposed to relatively lower pressure. Furthermore, it has been known that the high-pressure treatment induces fragmentation of myofibrils, modification of the actin-myosin interaction and activation of intramuscular proteinase, cathepsins and calpains. Thus, proteasome probably is implicated in the tenderization process in combination with other intramuscular proteinase under the high-pressure condition. From all the results obtained in the present experiments and in other investigators'reports, it is presumed that proteasome may play a role in meat tenderization during aging or by high-pressure treatment