We have previously demonstrated that ischemic injury results in the loss of peroxisomal functions (e.g., inhibition of
catalase activity and
fatty-acid beta-oxidation activity). To understand the molecular mechanism leading to the loss of peroxisomal beta-oxidation in ischemic tissue, we examined the levels of individual
enzyme activities and
proteins of the peroxisomal beta-oxidation system and overall
fatty-acid oxidation in peroxisomes isolated from kidney exposed to
ischemia-reperfusion injury. The peroxisomal beta-oxidation decreased with an increase in time of ischemic injury (53% and 43% of the control in kidneys exposed to 60 and 90 min
ischemia, respectively). In vivo inactivation of
catalase with
aminotriazole and exposure of isolated peroxisomes to H2O2 resulted in inhibition of peroxisomal beta-oxidation system suggesting that this
enzyme system is labile to excessive H2O2 produced during ischemic injury. The
enzyme activities of
lignoceroyl-CoA ligase,
acyl-CoA oxidase, bifunctional
enzymes and
acyl-CoA thiolase (individual peroxisomal beta-oxidation
enzymes) after 90 min of
ischemia were 87, 80, 87 and 85% of the control, respectively. This decrease in
enzyme activities was more pronounced following reperfusion (28, 11, 23 and 35% of the control, respectively). Immunoblot analysis of these
enzymes indicated that the major loss of these
enzyme activities during
ischemia was due to their inactivation, whereas during reperfusion, proteolysis also contributed toward the observed loss of these activities. In summary, these results demonstrated that loss of peroxisomal beta-oxidation in
ischemia-reperfusion injury was due to inactivation and proteolysis of beta-oxidation
enzymes.
Acyl-CoA oxidase was more sensitive to
ischemia-reperfusion injury compared to other
enzymes, and the overall loss of peroxisomal beta-oxidation may be a reflection of the loss of
acyl-CoA oxidase activity, a rate-limiting
enzyme.