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. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach.

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.

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.

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies.

BACKGROUND: Proteasomes are nonlysosomal proteolytic structures that have been implicated in cell growth and differentiation. Abnormal expression levels of proteasomes have been described in tumor cells, and proteasomes can be detected and measured in plasma. The objective of this study was to characterize differences in proteasome levels between normal, healthy donors and patients with neoplastic disease and to correlate the findings with clinical status and other biologic markers of disease spread. METHODS: Plasma proteasome levels were measured using a sandwich enzyme-linked immunosorbent assay in normal donors (n = 73 donors) and in patients with solid tumors (n = 20 patients), acute leukemia (n = 35 patients), myeloproliferative (n = 37 patients) and myelodysplastic (n = 19 patients) syndromes, chronic lymphocytic leukemia (n = 44 patients), non-Hodgkin lymphoma (n =104 patients), Hodgkin disease (n = 14 patients), other lymphoid disorders (n = 17 patients), and multiple myeloma (n = 27 patients). RESULTS: In the normal donors, the plasma proteasome concentration was 2356 ng/mL +/- 127 ng/mL. Patients with solid tumors exhibited a significantly higher value (7589 ng/mL +/- 2124 ng/mL), similar to the patients with myeloproliferative (4099 ng/mL +/- 498 ng/mL) and myelodysplastic (2922 ng/mL +/- 322 ng/mL) syndromes. Patients with lymphoproliferative disorders, in contrast, had significantly lower values than normal donors (1751 ng/mL +/- 107 ng/mL), except those in aggressive phase of the disease. This low level persisted in patients who were in complete remission. Proteasome levels decreased during the initial phase of treatment. Although there was a significant correlation with serum lactic dehydrogenase levels, frequent discrepancies were noted. There was no correlation with C-reactive protein or beta2-microglobulin levels, even in the group of patients with multiple myeloma. CONCLUSIONS: The plasma proteasome level is a potential new tool for the monitoring of patients with neoplastic disease. It is not correlated solely with cell lysis and may be involved in the pathophysiology of disease progression.

Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact!

Myoglobin has been suggested to be a potential inhibitor of endogenous muscle proteases as different as cathepsin B, cathepsin L, cathepsin H and calpains all being supposed to be important in post-mortem muscle. The present work aimed at verifying the ability of myoglobin and its prosthetic group, hemin, to inhibit a series of endopeptidases including papain, cathepsin B, trypsin, calpains as well as two activities of the 20S proteasome. The conclusion of the present work was that inhibition of proteolytic activities of endopeptidases by myoglobin is an artifact. This was based on the following evidences: (1) a similar extent of inhibition was observed for all proteases tested whether myoglobin or hemin were added before starting the reaction or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assayed with non labeled casein as substrate and the activity expressed as the increase in the absorbency at 280 nm of the TCA soluble protein fragments.(1).

Isolation and identification of a mu-calpain-protein kinase C alpha complex in skeletal muscle.

A mu-calpain-PKC complex was isolated from rabbit skeletal muscle by ultracentrifugation and by anion-exchange chromatography. The PKC associated to mu-calpain was stimulated by calcium, phosphatidylserine and diacylglycerol, and corresponds to a conventional PKC (cPKC). This complex presents an apparent molecular mass close to 190 kDa and is composed of one mu-calpain molecule and of one cPKC molecule. Using monoclonal antibodies specific for the different cPKC isoforms, the isoenzyme associated to mu-calpain was identified as cPKC alpha. Immunofluorescence staining reveals a co-localization of mu-calpain and cPKC alpha on the muscle fibre plasma membranes.