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The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria.

ABSTRACT: 1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate-cytochrome c oxidoreductase and NADH-cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP(+)-linked) and NADPH-cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD(+)- and NADP(+)-linked), alpha-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD(+)-linked), glutamate-oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, alpha-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD(+)- and NADP(+)-linked), glutamate dehydrogenase (NAD(+)-linked), glutamate-oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.

SUBMITTER: Chapman C

PROVIDER: S-EPMC1198688 | biostudies-other | 1968 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundThe mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.ResultsNAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.ConclusionLoss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.

Project description:Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

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The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria.

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