Chen G X, Mueller C, Wendlinger M, Zolg J W
Mol Pharmacol. 1987 Apr;31(4):430-7.
Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+-oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min-1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg-1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500-fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.
通过亲和层析法从人类疟原虫恶性疟原虫的三个克隆株中部分纯化了二氢叶酸还原酶（DHFR）（5,6,7,8-四氢叶酸：NADPH + -氧化还原酶；EC 1.5.1.3）。这三个克隆株分别代表对乙胺嘧啶敏感的（克隆株3D7）和对乙胺嘧啶耐药的（克隆株HB3和克隆株7G8）寄生虫，在体外对该药物进行测试时，其ID50值分别为0.53 nM（3D7）、210 nM（HB3）和540 nM（7G8）。在敏感克隆株3D7（442±39 nmol min-1 mg-1蛋白质）与耐药克隆株HB3（634±25 nmol min-1 mg-1蛋白质）和7G8（565±85 nmol min-1 mg-1蛋白质）之间，部分纯化的DHFR的比活性差异小于2倍。来自三个克隆株的部分纯化的DHFR中的催化位点数量相似，范围为151至194 pmol mg-1蛋白质。所有三个克隆株中NADPH的Km值相似（4.5 - 11.6 microM）。将敏感克隆株3D7（3.2±0.6 microM）与耐药克隆株HB3（42.6±1.6 microM）相比，二氢叶酸的Km值改变了13倍，耐药克隆株7G8的Km值介于两者之间（11.9±1.2 microM）。乙胺嘧啶的抑制常数从0.19±0.08 nM（3D7）增加到2.0±0.3 nM（HB3）再增加到8.9±0.8 nM（7G8）。乙胺嘧啶对敏感克隆株3D7的抑制是非竞争性的，而对另外两个克隆株是竞争性的。用乙胺嘧啶滴定部分纯化的DHFR发现，当将敏感克隆株3D7（0.18±0.02 nM）与耐药克隆株7G8（95±16 nM）相比时，抑制DHFR活性50%所需的药物浓度增加了500倍。通过比较比活性和催化中心活性以及底物的Km值和乙胺嘧啶的抑制常数（这两者在耐药克隆株中均发生改变），我们得出结论，所研究的三个克隆株中乙胺嘧啶耐药的分子机制不是基于DHFR的过量产生，而是由于结构改变的酶对抗叶酸药物的亲和力降低。
