A localized
burn injury to a rat hindlimb was used to investigate the
proteolytic enzymes responsible for the
burn-induced increase in
muscle protein breakdown. In 10,000 x g pellets of muscle homogenates,
burn stimulated (50% to 100%)
protease activities with pH optima of 3, 5.5, and 7.8.
Burn also stimulated
acid protease activity in 27,000 x g supernatants derived from
triton X100 treated extracts of muscle. Pretreatment of rats with
compound 48/80 (to eliminate contaminating mast cells) eliminated 95% of the neutral
protease activity in the particulate fraction. The Ca++-sensitive neutral
protease was not affected by either
burn or 48/80 treatment. However, muscle extracts from the burned leg always showed a 40% to 70% increase in
acid protease activity. All of the
acid protease activity could be inhibited by a combination of
cathepsin inhibitors
pepstatin (0.01 microgram/mL) and
leupeptin (1 mumol/L) and
leupeptin (1 mumol/L) or Ep475 (1 microgram/mL).
Leupeptin and the lysosomotropic agent
leucine methyl ester also inhibited the
burn-induced proteolysis in intact muscle. A time course shows parallel increases in whole muscle proteolysis and
acid protease activity of muscle homogenates. These findings support the conclusion that the increase in lysosomal
cathepsins are sufficient to account for the
burn-induced increase in
protein breakdown in muscles from the injured leg.