Glutathione-S-transferases from chloroquine-resistant and -sensitive strains of Plasmodium falciparum: what are their differences?

Glutathione-S-transferases (GSTs) from chloroquine-resistant (CQR, K1) and -sensitive (CQS, T9/94) strains of Plasmodiumfalciparum were studied. The enzymes from both strains exhibited the optimal pH for enzyme catalysis, at pH 7.5, and were stable at temperatures below 60 degrees C. They also showed the highest specific activities toward CDNB and moderate activities to ethacrynic acid (40% of the activity to CDNB) but little or no activity for other substrates. Km and Vmax values for CDNB and GSH, calculated by Lineweaver-Burk plot from both CQR- and CQS-GSTs, were not statistically different (p<0.05). However, the GSTs activity from CQR appeared to be significantly higher than that from CQS. Therefore, we proposed that GSTs from both malarial strains are identical in their functional domain but different in level of gene expression. Furthermore, protein sequence alignment between P. falciparum GST and GSTs from other organisms suggested that the malarial enzyme is closely similar to other GSTs in Sigma, Alpha, Mu and Pi subclasses, probably most to the Alpha group. Characterization of the purified malarial GST in detail would reveal more precise classification and better understanding of its role in malarial detoxification.

Partial purification of mitochondrial DNA topoisomerase II from Plasmodium falciparum and its sensitivity to inhibitors.

Mitochondria of Plasmodium falciparum (K1 strain) were isolated by differential centrifugation. Mitochondrial DNA topoisomerase II from P. falciparum was partially purified using fast protein liquid chromatography(FPLC). Parasite mitochondria contained approximately 8% of DNA topoisomerase II activity compared with its nuclear fraction. The effects of fluoroquinolones, inhibitors of bacterial DNA topoisomerase II or DNA gyrase, against partially purified P. falciparum mitochondrial DNA topoisomerase II were investigated using a decatenation assay. Minimum inhibitory concentrations (MIC) of ofloxacin, ciprofloxacin and norfloxacin were > 1, 10 and 100 mM, compared with that of >0.5 and 10 mM for eukaryotic DNA topoisomerase II inhibitor etoposide (VP-16) and amsacrine, respectively. The results indicate that partially purified mitochondrial DNA topoisomerase II was insensitive to fluoroquinolones and it is suggested that their inhibitory effects on P. falciparum growth may be directed against plastid DNA topoisomerase II.