Membrane-deoxyribonucleic acid (DNA) complexes were isolated from Bacillus subtilis by affinity for magnesium-Sarkosyl crystals. These complexes (Mbands) contained greater than 80% of the total cellular DNA; little of the remaining portion could be recovered in a secondary isolation. Isotopic labeling of the origin of replication showed this region of the chromosome to be closely associated with the cell membrane. Interruption ofprotein or DNA synthesis did not result in detachment ofthe chromosome from the membrane. Interruption of ribonucleic acid synthesis by rifampin resulted in a decreased ability to isolate DNA in the M-band. Analysis of attachment of the chromosome to membrane during the cell and replication cycles indicated that the chromosome is not released from the membrane at any time during the cell cycle.
The in vitro toxicity of multiple hydrophobic compounds was the focus of this study. A mitochondrial respiratory assay, sensitive to perturbations caused by hydrophobic chemicals, was utilized to measure the effects of individual aromatic hydrocarbon pollutants and their mixtures on mitochondrial respiratory function. Benzene, naphthalene, acenaphthene, and 1-chloronaphthalene, common industrial solvents shown to interact additively in vivo, were evaluated using this in vitro assay system. Mitochondrial respiration was inhibited 50% (EC50) by 525 ppm (6.7 mM) benzene, 15 ppm (117 microM) naphthalene, 3.9 ppm (25.5 microM) acenaphthene, or 3.8 ppm (23.4 microM) 1-chloronaphthalene. NADH:O2 oxidoreductase (NADH-->O2), NADH:ubiquinone oxidoreductase, and ubiquinol:O2 oxidoreductase activities were inhibited by all four compounds, whereas succinate:O2 oxidoreductase, cytochrome c oxidase, and duroquinol:O2 oxidoreductase activities were not inhibited. Inhibition of mitochondrial respiration occurred at the level of ubiquinone (coenzyme Q10) for all four aromatic hydrocarbons. The ultraviolet absorbance spectrum of isolated Q10 was also altered by naphthalene, acenaphthene, or 1-chloronaphthalene, suggesting a specific interaction between that component of the respiratory chain and these aromatic hydrocarbons. Inhibition by a mixture of 2, 3, or 4 of the compounds tested was additive, reflecting a summation effect of each compound present in the mixture. This additive nature is consistent with previously reported effects of these compounds in vivo and with compounds having similar modes of action. The similar mode of action in vitro is a specific interaction with coenzyme Q10, not a generalized membrane perturbation as speculated to occur in vivo, and is the likely mechanism for the observed additive toxicity.
Membrane-deoxyribonucleic acid complexes (M-bands) have been isolated from Bacillus subtilis by their affinity for crystals of Mg2+-Sarkosyl. The membrane proteins of these complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the membrane protein composition of M-band and unfractionated membrane revealed three protein components of 125,000 (mac-1), 57,000 (mac-2), and 42,000 (mac-3) daltons unique to M-band membrane. Growth of a temperature-sensitive dna initiation mutant at the restrictive temperature resulted in an accumulation in the membrane of mac-2. This accumulation did not begin, however, until cell growth had nearly ceased, some 3 to 4 h after the cessation of deoxyribonucleic acid synthesis. Upon return of the mutant to the permissive temperature, mac-2 did not begin to return to normal levels until after the first round of deoxyribonucleic acid synthesis. A protein of 30,000 daltons, common to both M-band and whole membrane, was found to disappear from the membrane when the mutant was grown at the restrictive temperature. This disappearance is the result of increased degradation or removal from the membrane followed by a decreased rate of synthesis or insertion.
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