Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria.
A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.
Lazarus
G M
GM
Department of Chemistry, University of Maryland Baltimore County, Catonsville 21228.
Henrich
J P
JP
Kelly
W G
WG
Schmitz
S A
SA
Castora
F J
FJ
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D013367:Submitochondrial Particles / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology*
D004264:DNA Topoisomerases, Type I