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The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.z 1999 Federation of European Biochemical Societies.

A human milk fraction containing multimeric a-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of a-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of a-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (DC m ), mitochondrial swelling and the release of cytochrome c. These changes were Ca 21 -dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of DC m , indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human a-lactalbumin made lethal to tumour cells), which is native a-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of a-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.Keywords: a-lactalbumin; cytochrome c; mitochondria; mitochondrial permeability transition; tumour cells.Diverse physiological and pathological stimuli can induce apoptosis by distinct pathways that converge in a common programme of cell suicide. Upon apoptotic triggering, a unique family of cysteine aspartate proteases, caspases, becomes activated [1]. In many models of apoptosis, caspase activation requires the release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space. Once released into the cytosol, cytochrome c binds to Apaf-1, and together with pro-caspase-9 and dATP, forms the so-called apoptosome complex [2]. This association leads to the activation of pro-caspase-9 which, in turn, initiates the caspase cascade by activating pro-caspase-3. Although cytochrome c release has been observed in many experimental models of apoptosis, the precise mechanism responsible for its movement across the outer mitochondrial membrane remains unclear. Different hypotheses have been proposed [3]. Cytochrome c may be released via specific channels in the outer mitochondrial membrane or the release may be a consequence of mitochondrial swelling leading to the rupture of the outer mitochondrial membrane. Mitochondrial swelling may be due to opening of the mitochondrial permeability transition (MPT) pore, a polyprotein complex fo...

To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.