DNA topoisomerase I from calf thymus mitochondria is associated with a DNA binding, inner membrane protein.
During purification of the type I DNA topoisomerase from calf thymus mitochondria, two polypeptides, p78 and p63, cofractionate with the enzymatic activity (Lazarus et al., (1987) Biochemistry 26, 6195-6203). The two polypeptides are released from a mitochondrial inner membrane preparation by nonionic detergent lysis and both adsorb strongly to a single-stranded DNA agarose column. We have attempted to characterize the relationship between these two polypeptides and have found the following: (i) the mitochondrial topoisomerase is active in free (monomer) and associated (heterodimer) form; (ii) the catalytic activity resides solely in p78, as adjudged by both the covalent linkage of the enzyme to substrate DNA and the ability of the enzyme to relax supercoils; (iii) at low ionic strength the enzyme is active in monomer form with p78 alone being sufficient for activity; (iv) in high salt, the high molecular weight species is a 140-kDa heterodimer composed of one p78 and one p63; and (v) the two polypeptides are not structurally related as digestion with V8 protease results in distinct proteolytic fragment patterns. These results suggest that p63 may have an important role in the metabolism of the mitochondrial topoisomerase.
Lin
J H
JH
Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23501.
Lazarus
G M
GM
Castora
F J
FJ
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007425:Intracellular Membranes / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry*; D008623:Mercaptoethanol / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D010455:Peptides; D008623:Mercaptoethanol; D004264:DNA Topoisomerases, Type I