Isamu Tani

Rhodamine 6G, inhibitor of both H+-ejections from mitochondria energized with ATP and with respiratory substrates.

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F1. Rhodamine 6G also inhibited both H+-ejections from mitochondria energized with respiratory substrates and with ATP. The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, Pi, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H+-ejection by redox components and also on F0.

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.

Stoichiometry of chargerin II (A6L) in the H+-ATP synthase of rat liver mitochondria

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the A6L of mitochondrial DNA, may have a key role in the energy transduction by mitochondrial H(+)-ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the contents of chargerin II in the H(+)-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H(+)-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H(+)-ATP synthase.

Orientation of chargerin II (A6L) in the ATP synthase of rat liver mitochondria determined with antibodies against peptides of the protein

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the unidentified reading frame A6L of mitochondrial DNA (URFA6L), may have a key role in the energy transduction by mitochondrial ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the orientation of chargerin II in Fo of the ATP synthase of rat liver mitochondria was examined using antibodies against peptides of chargerin II. Results showed that its N-terminal region (about 8 amino acid residues) was exposed on the surface of the C-side of Fo, but its C-terminal and charge-cluster regions were buried in Fo.

A hydrophobic protein, chargerin II, purified from rat liver mitochondria is encoded in the unidentified reading frame A6L of mitochondrial DNA.

Previous studies showed that a hydrophobic protein called chargerin II may have a key role in energy transduction of oxidative phosphorylation, since antibody against chargerin II labeled with monoazide ethidium inhibited ATP synthesis, ATP-Pi exchange, and reversed electron flow from succinate to NAD coupled with succinate oxidation by O2. In the present work, unlabeled chargerin II was purified from intact rat liver mitochondria by high performance liquid chromatography. The purified preparation of chargerin II, which was a single protein as judged by polyacrylamide gel electrophoresis and Western blotting, was digested with lysylendopeptidase. The digest was separated on a reverse-phase column into five peptides, which all cross-reacted with the antibody against chargerin II, indicating that they were fragments of chargerin II. The sequences of two of these peptides (a total of 12 amino acids) were determined and found to be highly homologous with the sequence of the carboxyl-terminal peptide of the putative polypeptide encoded by the unidentified reading frame A6L (URFA6L) of mammalian mitochondrial DNA. The amino acid compositions of the purified preparation of chargerin II were in good accord with those of the putative product of the URFA6L. Thus, we concluded that chargerin II is encoded by the URFA6L. This is the first demonstration that the URFA6L product was identified in rat liver mitochondria and purified from the membranes.

Laser Raman Spectroscopy of Lyophilized Bacterial Spores

Laser‐excited Raman spectra were examined in lyophilized spores of Bacillus cereus. In a comparison of the spectrum of the dormant spore with that of the germinated spore, we found several Raman bands which occurred in the former but not in the latter. Among these Raman bands, the 1,573, 1,395, 1,017, 822, and 662 cm−1 bands were assigned to the vibrational frequencies of calcium dipicolinate (CaDPA). No Raman bands and peaks due to dipicolinic acid (H2DPA) were observed. This Raman evidence indicates that CaDPA is the predominant DPA species in this spore.

Immunochemical study of role of chargerin II, a product of URFA6L of mitochondrial DNA in energy transduction of rat liver mitochondria

Antibody against chargerin II [product of the unidentified reading frame A6L (URFA6L) of mitochondrial DNA] inhibited the ATP-Pi exchange reaction and reversed electron flow from succinate to NAD in mitoplasts (inner membrane plus matrix). The antibody against chargerin II caused greater inhibition on incubation with mitoplasts in the energized than the nonenergized state, suggesting that redox reactions are coupled with a conformational change of chargerin II. The present findings showed that chargerin II, the URFA6L product, may have a key role in energy transduction of mitochondrial oxidative phosphorylation.

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 mug antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c1 completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.

Triphenyltetrazolium and its derivatives are anisotropic inhibitors of energy transduction in oxidative phosphorylation in rat liver mitochondria

Triphenyltetrazolium and its derivatives inhibited energy transduction in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. Triphenyltetrazolium incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. Triphenyltetrazolium also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced sites on the membranes for triphenyltetrazolium and its derivatives. The maximum amounts of energy-dependent binding sites for triphenyltetrazolium on membranes energized with succinate and ATP, respectively, were 14 and 4 nmol/mg protein. Triphenyltetrazolium also induced H+ ejection from the energized membranes. The maximum amounts of H+ ejection from membranes energized with succinate and ATP, respectively, were 4 and 2.4 nmol/mg protein. Triphenyltetrazolium also decreased the membrane potential up to about half the control value and caused shrinkage of mitochondria in an energy-dependent fashion. Comparison of the Hammetts sigma constants of triphenyltetrazolium derivatives with various substituents on the 3-benzene ring showed that lower concentrations of triphenyltetrazolium derivatives with a stronger positive charge were required for inhibition of energy transduction. The present findings show that triphenyltetrazolium and its derivatives act as anisotropic inhibitors of energy transduction by binding to negative charges created on the outer side (C-side) of energized mitochondria, and that the positive charge of these inhibitors is one of important factors for their inhibitory activity. These negative charges may be an essential part of the H+ pump.

Involvement of Calcium in Germination of Coat‐Modified Spores of Bacillus cereus T

The effect of calcium on germination of coat‐modified Bacillus cereus T spores was investigated. Coat‐modified spores produced either by chemical extraction (SDS‐DTT‐treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS‐DTT‐treated spores could germinate slowly in the presence of l‐alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat‐modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat‐modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat‐modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which l‐alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS‐DTT‐treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.