ETHANOL-CONSUMPTION ALTERS TRAFFICKING OF LYSOSOMAL-ENZYMES AND AFFECTS THE PROCESSING OF PROCATHEPSIN-L IN RAT-LIVER

In order to determine whether ethanol consumption alters the targeting of hepatic lysosomal enzymes to their organelles, we examined the sed imentation properties of lysosomal hydrolases in ethanol fed rats and their pair-fed controls. Rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose dextrin for one to fi ve wk. Liver extracts were fractionated by Percoll density gradient ce ntrifugation and fractions obtained were analyzed for the distribution of lysosomal marker enzymes. Heavy lysosomes were further purified fr om these gradients and the activity of specific hydrolases was determi ned. Compared with those from controls, isolated lysosomes from ethano l-fed rats showed a 20-50% reduction in the activity of lysosomal acid phosphatase and P-galactosidase. Decreased intralysosomal hydrolase a ctivity in ethanol-fed rats was associated with a significant redistri bution of these enzymes as well as those of cathepsins B and L to ligh ter fractions of Percoll density gradients. This indicated an ethanol- elicited shift of these enzymes to lower density cellular compartments . In order to determine whether ethanol administration affects the syn thesis and proteolytic maturation of hepatic procathepsin L, we conduc ted immunoblot analyses to quantify the steady-state levels of precurs or and mature forms of cathepsin L in hepatic post-nuclear fractions. Ethanol administration caused a significant elevation in the steady-st ate level of the 39 kDa cathepsin L precursor relative to its 30 kDa i ntermediate and 35 kDa mature product. These results were confirmed by pulse-chase experiments using isolated hepatocytes exposed to [S-35]m ethionine. Hepatocytes from both control and ethanol-fed rats incorpor ated equal levels of radioactivity into procathepsin L. However, durin g the chase period, the ratios of the 39 kDa procathepsin L to its 30 kDa intermediate and 25 kDa mature product in cells from ethanol-fed r ats were 1.5-3-fold higher than those in controls. These results demon strate that ethanol consumption caused a marked impairment in the proc essing of procathepsin L to mature enzyme, without affecting its synth esis. Taken together, our findings suggest that chronic ethanol consum ption caused a deficiency in intralysosomal enzyme content by altering the trafficking and processing of these hydrolases into lysosomes.