Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.     

Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications

The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331μg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213μg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209μg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203μg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the μg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.     

Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure

This paper describes the purification and properties of a multicatalytic proteinase complex, proteasome, from rabbit skeletal muscle, and its effect on myofibrillar structure. The purified proteasome gave a single band on polyacrylamide gel electrophoresis under non-denaturing conditions and gave eight bands under denaturing conditions, indicating that this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data indicate that the purified proteasome is not 26S, but 20S. The proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5.0 still retained about 30˜60% of the activity at pH 8.0, indicating the possibility that the proteasome is active in the muscle during conditioning. When the proteasome was heated at 60 °C for 20 min and treated in the presence of 0.005% SDS, the activity increased over 1.5 and 4.5 times, respectively. SDS remarkably increased the V_max value of the enzyme at pH 8.0. The proteasome was also activated by high hydrostatic pressure up to 100˜150 M Pa and gradually decreased at 200 MPa or higher. Electron microscopic observation revealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by proteasome.     

Structural studies of rigor bovine myofibrils using fluorescence microscopy. II. Influence of sarcomere length on the binding of myosin subfragment-1, alpha-actinin and G-actin to rigor myofibrils

Sarcomere length influences the textural quality of meat, yet the molecular basis for the mechanism of post-mortem shortening and the toughness associated with shortened muscle remain obscure. Bovine and rabbit myofibrillar structure was investigated over a range of sarcomere lengths (SL), using the high affinity of the myosin head for actin and of alpha-actinin for the Z-line. Myofibrils were incubated with fluorescent conjugates of myosin subfragment-1 (S1), alpha-actinin and actin. When S1 and alpha-actinin were added together, S1 bound in the I-band and A-band but not at the Z-line, while alpha-actinin bound at the Z-line. The pattern of S1 binding was highly influenced by SL, and could be explained using a model with the ratio of myofibrillar actin to myosin heads in the overlap region of 2:1 and thin filament penetration into opposing half sarcomeres. Double staining with S1 and actin demonstrated that, once the tip of the thin filament reached the bare zone, few intrinsic myosin heads were available for fluorescent actin. The patterns observed for both S1 and actin staining suggest that myofibrillar rigor bonds form even at very short SL. These observations lead to the hypothesis that the toughness associated with short sarcomeres is due to thick-filament interactions and not to the density of rigor bonds in the myofibril. Regulation of S1 binding was investigated by incubating myofibrils with low levels of fluorescent S1 in the presence and absence of calcium; S1 binding was in the overlap region when calcium was absent, but in the I-band when it was present. These results suggest that the thin filament can be activated for muscle shortening by the binding of myosin heads, a mechanism that may contribute to post-mortem muscle shortening.     

The effect of proteasome on myofibrillar structures in bovine skeletal muscle

When bovine myofibrils are incubated with the 20S proteasome their structure is rapidly damaged with loss of material, particularly from the Z discs and I bands. After 24 hr of incubation the myofibrils rupture and debris appears. Certain myofibrillar proteins, including nebulin, myosin, actin and tropomyosin, are hydrolysed during the incubation; others are solubilised (α-actinin). The 20S proteasome completely and rapidly hydrolyses purified myofibrillar proteins in an energy-independent manner. This shows that the 20S proteasome probably plays a role in the postmortem transformation of muscle and more generally in the hydrolysis of cellular proteins.(1).     

Biochemistry of postmortem muscle — Lessons on mechanisms of meat tenderization

It is certain that meat tenderness is a highly valued consumer trait and thus definition of the multiple processes that influence meat tenderness will provide clues toward improving meat quality and value. The natural process by which meat becomes tender is complex. Tenderness development is dependent on the architecture and the integrity of the skeletal muscle cell and on events that modify those proteins and their interaction. Specifically protein degradation and protein oxidation have been identified as processes that modify proteins as well as the tenderness of meat. The intracellular environment is a major factor that controls these events. Ultimately, the interplay between these events determines the rate and extent of tenderization. Given the intricacy of the structure of the muscle cell, coupled with the complexity of the regulation of protein modification and the ever-changing intracellular environment it is not surprising that this area of research is a very dynamic field. Just as the overall integrity and function of muscle cells does not depend on a single protein, but rather on the coordinated interaction of several proteins, the structural weakening of muscle cells during postmortem aging also must not depend on the degradation of a single myofibrillar or other cytoskeletal protein. The proteins mentioned in this review are located in different regions of the muscle cell, and most have been implicated in some manner as being important in maintaining the structure and function of the muscle cell. Oxidation of myosin heavy chain, a predominant protein in the myofibril, is known to promote aggregation and toughening of meat. Degradation of proteins such as desmin, filamin, dystrophin, and talin (all located at the periphery of the Z-line) may disrupt the lateral register and integrity of the myofibril themselves as well as the attachments of the peripheral layer of myofibril to the sarcolemma. Degradation of the proteins within the myofibril that are associated with the thick and thin filaments may allow lateral movement or breaks to occur within the sarcomeres of postmortem aged samples. Titin, nebulin, and troponin-T, by their ability to directly interact with, or modulate the interaction between, major proteins of the thick and thin filaments and (or) the Z-line, play key roles in muscle cell integrity. Disruption of these proteins, especially titin and nebulin, could initiate further physicochemical and structural changes that result in myofibril fragmentation and loss of muscle cell integrity, and ultimately in tenderization of the muscle. In order to make real progress in this area, the scientific community must have a global appreciation of how both the structural proteins and the key proteases are influenced by the vast changes that occur during the conversion of muscle to meat.     

Myofibrils of cooked meat are a continuum of gap filaments

When cooked meat is subjected to high degrees of stretch it becomes apparent in high magnification electron micrographs that A-filaments have ceased to exist. The A-band is filled with a coagulum of actomyosin. Fragmentation of this coagulum during stretch reveals an array of fine filaments (identified as gap filaments). This result is obtained irrespective of rigor temperature, state of contraction or degree of cooking. If the meat is first aged, the gap filaments surviving in the I-band are too weak to open up the A-band. The results show that myofibrils in cooked meat are entirely dependent on heat-stable gap filaments for structural continuity and tensile strength. Theories of meat tenderness must be revised accordingly.     

Contribution of major structural changes in myofibrils to rabbit meat tenderisation during ageing

The contribution of major structural (myofibrillar fragmentation upon mechanical treatment) and ultra-structural (Z-line degradation, loss of electron density of M-line, transversal disruption of sarcomeres at N(2)-line level, longitudinal fissure of myofibrils, and loss of transversal alignments of Z- and M-lines) changes in myofibrils to rabbit (Oryctolagus cuniculus L.) meat tenderisation, during the ageing period (9 days at +4?°C), was studied for different types of muscle (type I, semimembranosus proprius; type IIB, semimembranosus accessorius; and type IID, psoas major). The results strongly suggest that myofibrillar structure weakening at N(2)-line level (evaluated by myofibrillar fragmentation upon mechanical homogenisation and observed by transversal disruption of sarcomeres), which is very likely mediated by cysteine endopeptidases, might be the major structural change responsible for rabbit meat tenderisation during ageing. Both myofibrillar fragmentation and transversal disruption of sarcomeres are good ageing indices for rabbit meat. The other major ultra-structural changes in myofibrils appear to have no major role in rabbit meat tenderisation at refrigeration temperatures. Finally, it is proposed that meat tenderisation during ageing depends mainly on the specific cleavage of titin molecules/filaments and nebulin molecules, at their susceptible sites located at or very close to the N(2)-line region (extensible segment and near C-terminus, respectively), mediated by cysteine endopeptidases (possibly calpains).     

Role of the ubiquitin‐proteasome pathway on proteolytic activity in postmortem proteolysis and tenderisation of sheep skeletal muscle

The objective of this study was to investigate the effects of ubiquitin‐proteasome pathway on meat tenderisation. The sheep muscle longissimus lumborum was injected with or without PYR‐41 (inhibitor of ubiquitination) or MG‐132 (inhibitor of proteasome). Muscle samples were collected at 6, 15, 24 and 48 h after injection. Myofibrillar protein degradation, muscle ultrastructure and sarcomere length were determined. Results showed that inhibition of proteasome or ubiquitination affected sarcomere length at 48 h after treatments. Destruction of muscle ultrastructure in both treatments was reduced when compared to control. Inhibition of proteasome produced different fragments of myofibrillar proteins in comparison with control at 48 h. In conclusion, ubiquitin‐proteasome plays a role in postmortem proteolysis and might contribute to meat tenderisation.     

Pressure treatment of meat: Effects on thermal transitions and shear values

The effects of pressure treatment (150 MN m(-2) for 3 h at 0°C) on the pH, thermal transitions, ultrastructure and Warner-Bratzler shear values of post-rigor beef semimembranosus and longissimus dorsi muscles have been investigated. Pressure treatment resulted in a slight but significant increase in pH. Differential scanning calorimetry revealed large changes in the thermograms of muscle samples as a result of pressure treatment, in particular a transition attributed to F-actin was absent in the pressure-treated sample. Examination of the ultrastructure also revealed extensive change as a result of pressure treatment, particularly in the I-band and M-line region. Pressure treatment either did not change shear values or increased them, according to whether the muscle was in the stretched or contracted state, respectively. The results are thought to support a theory for contraction state toughness proposed by Voyle (1969) in which increasing toughness is caused by an increasing incidence of sarcomeres in which thick filaments have been compressed onto the Z-line, thus removing the I-band as a zone of weakness.     

A RESEARCH NOTE EFFECTS OF PRESSURIZATION AND REFRIGERATED STORAGE ON MICROSTRUCTURE OF FISH MUSCLE TISSUE

Fish muscle tissues were subjected to high hydrostatic pressures in the range of 1,000 - 3,000 atm and the tissues subsequently observed using transmission electron microscopy. Pressurization at 1,000 atm caused contraction of the sarcomere with the I-band thin filaments being lost while the A-band was unaffected. On pressurizing the tissues at 2,000 atm, the I-band region became more visible with breakdown of the thin filaments in this region as well as the Z-disks. There was gross disintegration ofmyofibrillar structure with collapse of A-band structure and T-tubular system on subjecting muscle tissue to 3,000 atm of pressure. However, there were no observable changes in myofibrillar structure beyond these initial changes during the subsequent 21 days of storage under refrigerated conditions (4–7C). The results provide the structural basis for pressure-induced changes in fish texture.     

Effect of high pressure on moisture and NaCl diffusion into turkey breast

High-pressure processing (HPP) affects food component diffusion. This study evaluated pressure (0.1-300 MPa) effect on the diffusion of NaCl and water into turkey breast at 25 °C. NaCl and water diffusion were found to be a function of pressure and holding time. During the pressure come-up-time, diffusion of water and NaCl into the sample was enhanced as compared to control. The diffusion coefficient of moisture infusing out of the sample was found to be a minimum at 150 MPa. The diffusion coefficient of NaCl infusing into the sample was maximum at 150 MPa. Within the range of experimental conditions studied, 150 MPa treatment (up to 15 min holding time) yielded meat samples with minimum hardness, gumminess, and chewiness. Ultrastructure images revealed that 150 MPa treatments caused swelling of myofibrils, disappearance of the M-line, reduced difference in the density of the A-band and I-band and breaking of segments of Z-line. HPP could be a useful technique for the salting of turkey meat.     

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.     

Sperm proteasome and fertilization

The omnipresent ubiquitin-proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.     

Meat tenderization by calcium chloride after osmotic dehydration

In this study, meat was tenderized by dipping the meat in a calcium-containing solution after osmotic dehydration. Dehydrated meats were dipped in a 150 mM calcium chloride solution for 3 h, and after that stored for 24, 48, and 168 h at 3 ～ 4°C. Then, meat was subjected to texture measurement, sensory evaluations, biochemical analysis and histological observations. A gradual decrease in the firmness and higher tenderness sensory scores were observed in the meat treated with calcium chloride as compared with the untreated meat. Few differences were observed in water holding capacity by treatment at any stage of storage. A gradual increase in the myofibril fragmentation from the calcium chloride-treated meats was observed throughout the storage compared with the controls. Broadening of the I band and a disordered Z-line were observed in the treated meat. The deformation of the honeycomb-like structure of the endomysium was also observed in the treated samples. Therefore, the dipping in 150 mM calcium chloride solution after dehydration can be applied to improve meat tenderness without detrimental effects on other palatability and quality traits.     

Effects of heat on meat proteins - Implications on structure and quality of meat products

Globular and fibrous proteins are compared with regard to structural behaviour on heating, where the former expands and the latter contracts. The meat protein composition and structure is briefly described. The behaviour of the different meat proteins on heating is discussed. Most of the sarcoplasmic proteins aggregate between 40 and 60 °C, but for some of them the coagulation can extend up to 90°C. For myofibrillar proteins in solution unfolding starts at 30-32°C, followed by protein-protein association at 36-40°C and subsequent gelation at 45-50°C (conc.>0.5% by weight). At temperatures between 53 and 63°C the collagen denaturation occurs, followed by collagen fibre shrinkage. If the collagen fibres are not stabilised by heat-resistant intermolecular bonds, it dissolves and forms gelatine on further heating. The structural changes on cooking in whole meat and comminuted meat products, and the alterations in water-holding and texture of the meat product that it leads to, are then discussed.     

Development in myofibrillar water distribution of two pork qualities during 10-month freezer storage

The effects of fresh meat quality (PSE versus DFD), freezing temperature (-20°C versus -80C°) and duration of freezer storage on changes in water mobility and distribution were followed at intervals of 1-2 months during 10-month freezer storage of pork using low-field NMR T(2) relaxometry. Fresh meat quality was found to have a strong significant effect (P<0.0001) on the amount of loosely bound water (relaxation time >100ms) also after freezing, which was reflected in a significantly lower cooking yield in PSE meat compared with DFD meat (P<0.0001). While no significant changes in the cooking yield were observed with increasing length of freezer storage, NMR T(2) relaxation measurements revealed a significant increase in the amount of loosely bound water in PSE meat with increasing length of freezer storage. This finding indicates that NMR T(2) relaxation measurements are quite sensitive to freezing-induced changes in the meat structure, causing a shift in the distribution of water and possibly capable of detecting these before they are reflected in a reduced cooking yield. In addition, an interaction between fresh meat quality and effect of length of freezer storage on the amount of very mobile water easily lost as drip was observed, implying that PSE meat is more susceptible to freezer storage-induced deteriorative changes in the meat structure, causing a shift in the distribution of water, than DFD meat.     

The non-sliding filaments of the sarcomere

A model is proposed for the 'gap' or 'third' filaments of muscle, here renamed 'T-filaments'. These consist of single titin molecules, spanning the half sarcomere from M-line to Z-line. Of several possibilities for stoichiometry and arrangement, the one most favoured here consists of six T-filaments lying longitudinally on the surface of the A-filament, one against each peripheral subfilament of myosin. C-protein molecules over-lie the T-filaments in transverse and axial rows, with their long axes following a helix. Each C-protein molecule helps to bind a pair of T-filaments to one A-strand (but not to bind A-strands together), and hinders the immune response of titin in the C-zone. The gap filaments arise from the coalescence of T-filaments in the I-band, and their extreme extensibility from an unravelling of a beaded structure in the titin molecule. A layout is presented for the molecule in zones of configuration and function. Possible arrangements of the T-filaments in the M- and N(2)-zones are discussed.     

Molecular Properties of Post-Mortem Muscle. 3. Electron Microscopy of Myofibrils

SUMMARY— A study was made of the fine structure of myofibril suspensions prepared from seven heifers immediately after death and after various times post-mortem. Studies on myofibrils sampled immediately after death showed that sucrose isolation gave the best structural preservation as indicated by maintenance of Z-line structure. Although the appearance of resting muscle was maintained in both sucrose and KCI preparations, several myofibrils from the KCI-treated preparations showed stretched sarcomeres. Glycerol-treated myofibrils usually had shorter sarcomere lengths than myofibrils prepared with the other two solvents. Although fibrillar preservation seemed adequate when glycerol was used, Z-line structure was seldom well-preserved with glycerol.
Myofibrils from muscle sampled 24 hr post-mortem at 2°C were supercontracted with thick filaments pushed against or through the Z-line, and no trace of l-bands remained. Myofibrils from muscle sampled 24 hr post-mortem at 16°C were contracted, but to a much lesser extent than 2°C-24 hr myofibrils. Storage at 2°C for 312 hr after death resulted in myofibrils that were contracted and that were structurally in a much poorer state of preservation than their 16°C counterparts. The 16°C-312 hr myofibrils were slightly contracted as indicated by the absence of H-zones and the presence of prominent, although narrowed, I-bands. All observations showed that shortening accompanying rigor mortis caused changes in banding patterns similar, and probably identical, to those predicted by Huxley's sliding filament model for contracting muscle.