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

Abstract

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.