We previously reported that oxidative stress is associated with 
unloading-mediated ubiquitination of muscle proteins. To further elucidate the 
involvement of oxidative stress in ubiquitination, we examined the 
ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen 
peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low 
molecular masses (less than 60 kDa) as well as high molecular masses (more than 
160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated 
and immediately disappeared after the treatment. Microsequencing revealed that 
the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate 
dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the 
mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. 
Under unloading conditions, such as tail-suspension and spaceflight, 
mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, 
E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated 
mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, 
while they did not affect the amount of poly-ubiquitinated LDH. In contrast, 
epoxomicin, a potent proteasome inhibitor, did not change the amount of 
mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it 
significantly increased the amount of poly-ubiquitinated LDH. Our results 
suggest that oxidative stress induces not only poly-ubiquitination but also 
mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation 
during unloading.