Clemens Broger

The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H2O2: A Spin-trapping/ESR and absorption spectroscopy study

H2O2 is catalytically metabolized by ferric lactoperoxidase (LPO)----compound (cpd) I----cpd II----ferric LPO cycles. An excess of the substrate, however, is degraded by a ferric LPO----cpd I----cpd II----cpd III----ferrous LPO----ferric LPO cycle. This latter pathway leads to the partial or total irreversible inactivation of the enzyme depending on the excess of H2O2 (H. Jenzer, W. Jones, and H. Kohler (1986) J. Biol. Chem. 261, 15550-15556). Spin-trapping/ESR data indicate that in the course of the reaction superoxide (HO2./O2-) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-1-pyrroline-N-oxide) desferrioxamine, albumin, or mannitol, do not prevent enzyme inactivation, we conclude that OH. generation is a site-specific reaction at or near the active center of LPO where bulky scavenger molecules may not be able to penetrate. We suggest the formation of OH. by a Fenton-like reaction between H2O2 and the intermediate ferrous state of the enzyme, which substitutes for Fe2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H2O2 to produce HO2. and H2O, or the prosthetic group to give rise to oxidative cleavage of the porphyrin ring structure of the heme moiety of LPO and thus to the liberation of iron.

Extraction, partial purification and functional reconstitution of two mitochondrial carriers transporting keto acids: 2-oxoglutarate and pyruvate

Bovine heart submitochondrial particles were treated with a medium containing Triton X‐114 and cardiolipin. The extract was subjected to hydroxyapatite chromatography. Only a few major polypeptides of similar molecular masses were found in the eluate, as shown by electrophoresis in an SDS‐polyacrylamide gel stained with silver. The eluate was reconstituted into liposomes and was shown to catalyse two different transport activities: 2‐oxoglutarate‐2‐oxoglutarate exchange sensitive to phthalonate and phenylsuccinate and pyruvate‐pyruvate exchange sensitive to 2‐cyano‐4‐hydroxycinnamate. Since both activities were found to have characteristics similar to those described for intact mitochondria, it was concluded that at least two of the polypeptides found in the hydroxyapatite eluate correspond to the two mitochondrial carriers.

Affinity chromatography purification of cytochrome c oxidase: Use of a yeast cytochrome c—thiol-sepharose 4B column

Cytochrome c oxidase (EC 19.3 .l) is a membranebound enzyme which catalyses the reduction of molecular oxygen to water. The reducing equivalents for this reaction are provided by the natural substrate of the enzyme, ferrocytochrome c. Cytochromes c have largely conservative structures, suggesting that they are descendants pf a common ancestor and have analogous functions [ 11. They form reversible associations with their reductases and oxidases, that are mediated by complementary charge interactions [2,3]. Separation of cytochrome c oxidase by affinity chromatography on a cytochrome c column has been attempted [4] although the technique proved to be effective only with the Neurospora enzyme [5]. The inability of a Sepharose 4B-cytochrome c column to easily and reproducibly separate cytochrome c oxidase may be due to the fact that the lysine residues, through which cytochrome c crosslinks to CNBractivated Sepharose 4B, are most probably the residues which are necessary for the cytochrome c oxidase and reductase binding [2,3,6]. Here we describe a technique which allows cytochrome c oxidase purification in one step ,starting from a mitochondrial Triton X-l 00 extract. It is based on the use of Saccharomyces cerevisiae cytochrome c which, through its cysteine residue located close to the N-terminus [ 1 ] be covalently linked a thiol-activated column, thus important lysine free binding cytochrome c oxidase reductase.

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Affinity chromatography purification of cytochrome c oxidase and b-c1 complex from beef heart mitochondria. Use of thiol-sepharose-bound Saccharomyces cerevisiae cytochrome c☆

A method for simultaneous purification of cytochrome c reductase and cytochrome c oxidase using a cytochrome c affinity column is presented. Cytochrome c from Saccharomyces cerevisiae was linked to an activated thiol-Sepharose gel via its Cys-102 residue located far from the lysine residues on the front side of the molecule, responsible for the interaction with the reductase and oxidase. In previously reported affinity chromatography techniques these lysine residues most probably reacted with the column. Cytochrome c oxidase and reductase from bovine heart mitochondria bind specifically to the affinity column and can be recovered separately at different ionic strength in the elution buffer. The enzymes are highly pure and active.

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain.

The binding of cytochrome c to the cytochrome bc1 complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc1 complex were measured. Mixtures of bc1 complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dual-wavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 12 derivative produced a diminution of the polypeptide of the bc1 coplex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c1, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc1 complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N′-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H+ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.

Isolation and Functional Reconstitution of the Dicarboxylate Carrier from Bovine Liver Mitochondria

In order to understand the structure and function relationship of mitochondrial transporters, recent efforts have been focused on isolation of the individual carriers and their functional reconstitution into proteoliposomes (for review see Nalecz , 1986). In line with these studies we reported a successful purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria (Szewczyk et al., 1987). In general, heart mitochondria are considered optimal for studies on purification of the inner membrane proteins. This comes from the fact that the cristae/matrix ratio of these organelles is exceptionally high (more membranes per unit of weight of the mitochondrial preparation) and, in addition, from that the activity of proteolytic enzymes in heart homogenates is relatively low. On the other hand, however, it is known that the mitochondrial dicarboxylate carrier activity is much higher in liver than in heart (Sluse et al., 1971). Although it has not been clarified whether this is due to difference in total amount of the carrier protein in mitochondria, we found that only very little of the translocator protein could actually be isolated from heart (Szewczyk et al., 1987). Thus it seemed logical to try to isolate the dicarboxylate carrier from liver mitochondria.

[6] Affinity chromatography purification of cytochrome-c oxidase from bovine heart mitochondria and other sources

Publisher Summary This chapter describes the affinity chromatography purification of cytochrome-c oxidase from bovine heart mitochondria and other sources. In the mitochondrial respiratory chain cytochrome- c oxidase catalyzes the electron transfer from ferrocytochrome c to molecular oxygen. Specific interaction between cytochrome- c oxidase and its electron donor is useful in developing an affinity chromatography purification technique for cytochrome- c oxidase based on cytochrome- c immobilized on a gel matrix. The solubilized proteins are loaded at low salt concentration to a column containing the gel and after washing the column, they are eluted by increasing the ionic strength of the buffer. Various detergents, mainly of the nonionic type, are used in the solubilization step and during the chromatography procedure. If the solubilization step is optimized and the protein of interest can be released from the membrane in a rather selective way, it is possible to purify it to homogeneity in one affinity chromatography step. The chapter describes cytochrome c gels that are used for several purifications.

[7] Resolution of cytochrome-c oxidase

Publisher Summary This chapter focuses on the resolution of cytochrome- c oxidase. Cytochrome- c oxidase from bovine heart mitochondria is a multisubunit complex consisting of 13 polypeptides and four prosthetic groups, two hemes and two coppers. Heme is present only in the complexes that contain the major three subunits of cytochrome- c oxidase. Subunits III do not contain heme, as the heme/protein ratio of cytochrome- c oxidase is increased by depletion of subunit III. The spectrum of free heme is different from the spectrum of the partially denatured sub-complexes, indicating that the heme is not associated to a denatured system. The heme-containing sub-complexes can be reduced and oxidized, and they exhibit CO binding capacity. Several methods are available to deplete cytochrome- c oxidase of its subunit III. The cytochrome c gel can be regenerated and used several times, although best yields and purity are obtained with freshly prepared gels, as it avoids the use of proteases and too high concentrations of detergents and is carded out mostly at physiological pH, thus ensuring that the enzyme remains as much as possible in its native state.