INCREASE IN UBIQUITIN PROTEIN CONJUGATES CONCOMITANT WITH THE INCREASE IN PROTEOLYSIS IN RAT SKELETAL-MUSCLE DURING STARVATION AND ATROPHY DENERVATION

The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakd own and activation of a non-lysosomal ATP-dependent proteolytic proces s. To investigate whether protein flux through the ubiquitin (Ub)-prot easome pathway is enhanced, as was suggested by related studies, we me asured, using specific polyclonal antibodies, the levels of Ub-conjuga ted proteins in normal and atrophying muscles. The content of these cr itical intermediates had increased 50-250% after food deprivation in t he extensor digitorum longus and soleus muscles 2 days after denervati on. Like rates of proteolysis, he amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively duri ng food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein con jugates than those of starved control animals. The changes in the pool s of Ub-conjugated proteins (the substrates for the 26S proteasome) th us coincided with and can account for the alterations in overall prote olysis. In this pathway, large multi-ubiquitinated proteins are prefer entially degraded, and the Ub-protein conjugates that accumulated in a trophying muscles were of high molecular mass (> 100 kDa). When innerv ated and denervated gastrocnemius muscles were fractionated, a signifi cant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denerva tion, but not in the soluble fraction. Thus activation of this proteol ytic pathway in atrophying muscles probably occurs initially by increa sing Ub conjugation to cell proteins. The resulting accumulation of Ub -protein conjugates suggests that their degradation by the 26S proteas ome complex subsequently becomes rate-limiting in these catabolic stat es.