Oxidative phosphorylation in a yeast mutant lacking α-ketoglutarate dehydrogenase activity

The effect of α-ketoglutarate deficiency on the oxidative phosphorylation in yeast mitochondria was studied. By determining the properties of electron transport and energy transduction systems of mutant mitochondria it was found that the lack of α-ketoglutarate dehydrogenase activity in mitochondria does not result in any functional defect in the oxidative phosphorylation system.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

Genetic determination of the mitochondrial adenine nucleotide translocation system and ITS role in the eukaryotic cell

On integrating experimental data published previously, the following picture of the mitochondrial adenine nucleotide (AdN) translocation system is being presented: 1. The AdN translocation system serves not only to transport ATP synthesized within mitochondria into the cytosol but also to transport cytosolic ATP into the mitochondria when oxidative phosphorylation is not functioning. 2. The AdN translocator is coded for by nuclear genes and the mitochondrial protein synthesis is not involved in its formation. 3. The AdN translocation system must be preserved and functioning even in cells which could dispense with oxidative phosphorylation. It assures appropriate concentrations of intramitochondrial ATP. 4. The intramitochondrial ATP is required for normal replication of mitochondrial DNA. Tis supports the view that the mitochondrion is a self-replicating semi-autonomous organelle. 5. The appropriate concentration of ATP must be present in mitochondria to make possible cell growth or multiplication. This points to a direct or indirect role of mitochondria in the control of cell proliferation.     

Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces different phenotypes.

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.     

A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.