We have reconstituted nucleosome core particles from reversed-phase-HPLC-purified chicken erythrocyte core histones and 145 bp random-sequence DNA fragments. Characterization of the resulting nucleoprotein complexes by sedimentation velocity, CD and DNase I footprinting showed that they are structurally indistinguishable from native nucleosome core particles. Furthermore, we have shown that the ability to reproduce these native-like structural features in these reconstituted nucleosome core particles is basically independent of the biological source or the method used (i.e. salt versus acid) for the extraction of histones before their HPLC fractionation. The usefulness and relevance of this approach for the reconstitution of native-like chromatin structures from histone types (histone variants/post-translationally modified histones), which are usually available only in relatively small amounts, is discussed.

The dithiol-reactive reagent phenylarsine oxide causes a pH-dependent stimulation of unidirectional K+ flux into respiring rat liver mitochondria. This stimulation is diminished by subsequent addition of either the dithiol 2,3-dimercaptopropanol or the monothiol 2-mercaptoethanol. In contrast, uncoupling by phenylarsine oxide is reversed by 2,3-dimercaptopropanol but not by 2-mercaptoethanol. The data suggest separate sites of interaction of phenylarsine oxide with mechanisms of K+ entry and ATP synthesis. Stimulatory effects of mersalyl and phenylarsine oxide on K+ influx are not additive. Thus PheASO and mersalyl may affect K+ influx at a common site. Pretreatment of the mitochondria with DCCD, which inhibits K+ influx, fails to alter sensitivity to PheAsO or mersalyl. Thus the DCCD binding site associated with the K+ influx mechanism appears to be separate from and independent of the sulfhydryl group(s) which mediate stimulation of K+ influx by PheAsO and mersalyl. PheAsO, like mersalyl, also increases the rate of unidirectional K+ efflux from respiring mitochondria. The combined presence of PheAsO plus mersalyl causes a greater stimulation of K+ efflux than is observed with either reagent alone.

Effects on Mg++ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K+ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K+ flux into respiring mitochondria, also stimulates Mg++ influx. The K+ analog Ba++, when taken up into the mitochondrial matrix, inhibits influx of both K+ and Mg++. The effect on Mg++ influx is seen only if Mg++, which blocks Ba++ accumulation, is added after a preincubation with Ba++. Thus the inhibition of Mg++ influx appears to require interaction of Ba++ at the matrix side of the inner mitochondrial membrane. Added Ba++ also diminishes observed rates of Mg++ efflux but not K+ efflux. This difference may relate to a higher concentration of Ba++ remaining in the medium in the presence of Mg++ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexyl-carbodiimide (DCCD), under conditions which result in an increase in the apparent Km for K+ of the K+ influx mechanism, results in inhibition of Mg++ influx from media containing approximately 0.2 mM Mg++. The inhibitory effect of DCCD on Mg++ influx is not seen at higher external Mg++ (0.8 mM). This dependence on cation concentration is similar to the dependence on K+ concentration of the inhibitory effect of DCCD on K+ influx. Although mitochondrial Mg++ and K+ transport mechanisms exhibit similar reagent sensitivities, whether Mg++ and K+ share common transport catalysis remains to be established.

No abstract