Proteasome from rabbit skeletal muscle: Some properties and effects on muscle proteins

Abstract

Rabbit proteasome, likely to be a 20S proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purified enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20000–29000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580 000–800 000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pH for hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) (Suc-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH 5–9 and up to 60 °C at pH 7.2. The enzyme activity was inhibited by diisopropyl fluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaCl 2. Remarkably, SDS increased the V max value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, α-actinin, actin, tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS.