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Peroxidative damage to cardiac mitochondria: identification and purification of modified adenine nucleotide
1Hormel Institute, University of Minnesota, Austin 55912.
Abstract:
Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118-123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu2+/t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.
Insights
Oxidative stress alters a key protein in rat heart mitochondria. This 28-kDa protein, identified as the adenine nucleotide translocase, is protected by carboxyatractyloside.
Area of Science:
- Biochemistry
- Cardiovascular Research
- Mitochondrial Biology
Background:
- Rat myocardial membranes undergo lipid peroxidation and protein alterations when exposed to free radicals.
- A specific 28-kDa polypeptide is highly sensitive, showing increased molecular weight before degradation.
Purpose of the Study:
- To identify and characterize the 28-kDa protein in cardiac mitochondria sensitive to oxidative damage.
- To investigate the role of the adenine nucleotide translocase in oxidative stress within the heart.
Main Methods:
- Treatment of isolated cardiac mitochondria with Cu2+/t-butylhydroperoxide.
- Analysis of protein alterations using molecular weight profiling.
- Utilizing carboxyatractyloside, an inhibitor of adenine nucleotide translocase, to assess its protective effects.
- Development of purification schemes for adenine nucleotide translocase.
Main Results:
- A 28-kDa protein in cardiac mitochondria exhibits sensitivity to Cu2+/t-butylhydroperoxide, mirroring previously observed alterations.
- This protein displays characteristics consistent with the mitochondrial adenine nucleotide translocase.
- Carboxyatractyloside effectively prevented the oxidative modification of the 28-kDa protein.
- Efficient methods were established for isolating both native and oxidatively modified adenine nucleotide translocase.
Conclusions:
- The mitochondrial adenine nucleotide translocase is a primary target of oxidative damage in rat heart mitochondria.
- Inhibition of adenine nucleotide translocase function protects it from oxidative modification.
- The study provides a method for isolating and studying this crucial mitochondrial protein under oxidative stress.