Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver.
A number of studies in yeast have shown that DNA topoisomerase II is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase II in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-free extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg(2+)-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.
Galande
S
S
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Muniyappa
K
K
D000818:Animals; D002417:Cattle; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D005971:Glutamates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D009928:Organ Specificity; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D013091:Spermatogenesis; D013737:Testis / Q000201:enzymology*; D013950:Thymus Gland / Q000201:enzymology
D004278:DNA, Superhelical; D005971:Glutamates; D007527:Isoenzymes; D004250:DNA Topoisomerases, Type II