ABSTRACT
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.
Subject(s)
Ischemia/physiopathology , Kidney/blood supply , Microbodies/metabolism , Reperfusion Injury/physiopathology , Acyl-CoA Oxidase , Amitrole/pharmacology , Animals , Carbon Radioisotopes , Catalase/metabolism , Electron Transport Complex IV/metabolism , Fatty Acids/metabolism , Hydrogen Peroxide/pharmacology , Male , NADH Dehydrogenase/metabolism , Oxidation-Reduction/drug effects , Oxidoreductases/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs.
Subject(s)
Cell Nucleus/enzymology , Peptide Hydrolases/isolation & purification , Zygote/enzymology , Animals , Chromatin/enzymology , Female , Histones/metabolism , Kinetics , Peptide Hydrolases/metabolism , Sea Urchins , Substrate SpecificityABSTRACT
1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.
