DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

Abstract

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [ 3H]dTTP or [ 3H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition. The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds. Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro. More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.