Donohue T M, Zetterman R K, Zhang-Gouillon Z Q, French S W
The Veterans Affairs Medical Center and the Department of Internal Medicine, University of Nebraska College of Medicine, Omaha 68105, USA.
Hepatology. 1998 Aug;28(2):486-91. doi: 10.1002/hep.510280228.
Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.
乙醇摄入会减缓肝脏蛋白质分解代谢的速率。本研究旨在确定通过自愿（配对）喂食或胃内给药方式给予乙醇，是否会影响大鼠肝脏中蛋白酶体的肽酶活性。将大鼠配对喂食含乙醇或等热量麦芽糖糊精的流质饮食。另一组动物通过胃内持续输注含乙醇或等热量葡萄糖的类似流质饮食。使用特定的荧光肽作为底物，对肝脏粗匀浆及其胞质部分进行胰凝乳蛋白酶样（Cht-L）、胰蛋白酶样（T-L）和肽基谷氨酰肽水解酶（PGPH）活性测定。自愿摄入乙醇并未影响蛋白酶体的三种肽酶活性。然而，胃内给予乙醇导致Cht-L和T-L活性下降35%至40%，但PGPH活性未显著改变。胃内给予乙醇的大鼠胞质样品中较低的肽酶活性，经过夜透析、在蛋白酶体测定混合物中加入低水平十二烷基硫酸钠（SDS）或0.5 mmol/L三磷酸腺苷（ATP）后，均未恢复到对照水平。使用抗大鼠肝脏蛋白酶体的免疫印迹分析显示，对照大鼠和乙醇喂养大鼠肝脏中免疫反应性蛋白酶体亚基水平相当。用专门针对蛋白酶体亚基LMP-7制备的抗体进行印迹检测时，也得到了类似结果。结果表明，胃内而非自愿摄入乙醇会差异性地影响蛋白酶体的不同催化活性，而不影响其稳态水平。这种变化可能与乙醇诱导的氧化应激程度有关。
