Kovacs J A, Allegra C J, Masur H
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland.
Exp Parasitol. 1990 Jul;71(1):60-8. doi: 10.1016/0014-4894(90)90008-z.
Pneumocystis carinii and Toxoplasma gondii are opportunistic pathogens of immunosuppressed patients that are susceptible to therapy with inhibitors of dihydrofolate reductase (DHFR). The DHFR of these two organisms was characterized to facilitate the identification of more selective inhibitors. Similar to all reported protozoa, T. gondii has a bifunctional enzyme, of 120,000 Da, that possesses both DHFR and thymidylate synthase (TS) activity. Unexpectedly, P. carinii DHFR activity was present on a small molecule, of 26,000 Da. T. gondii DHFR and TS activity coeluted during affinity chromatography using a methotrexate-Sepharose column, whereas P. carinii DHFR and TS activity could be separated by affinity chromatography using the same column. P. carinii DHFR could be easily distinguished from rat DHFR, which is similar in size, by the differences in Km for dihydrofolate (P. carinii, 17.6 +/- 3.9 microM; rat, 4.0 +/- 2.2 microM). Since all protozoa reported have a large molecular weight, bifunctional DHFR, these studies support the classification of P. carinii as a fungus. These studies also provide a basis for the development of more effective therapeutic agents for these pathogens.
卡氏肺孢子虫和刚地弓形虫是免疫抑制患者的机会性病原体，对二氢叶酸还原酶（DHFR）抑制剂治疗敏感。对这两种生物体的DHFR进行了表征，以促进更具选择性的抑制剂的鉴定。与所有已报道的原生动物类似，刚地弓形虫有一种120,000道尔顿的双功能酶，同时具有DHFR和胸苷酸合成酶（TS）活性。出乎意料的是，卡氏肺孢子虫的DHFR活性存在于一个26,000道尔顿的小分子上。使用甲氨蝶呤-琼脂糖柱进行亲和层析时，刚地弓形虫的DHFR和TS活性共同洗脱，而使用同一柱进行亲和层析时，卡氏肺孢子虫的DHFR和TS活性可以分离。卡氏肺孢子虫的DHFR与大小相似的大鼠DHFR很容易区分，这是由于它们对二氢叶酸的Km值不同（卡氏肺孢子虫为17.6±3.9微摩尔；大鼠为4.0±2.2微摩尔）。由于所有已报道的原生动物都有一个大分子量的双功能DHFR，这些研究支持将卡氏肺孢子虫归类为真菌。这些研究也为开发针对这些病原体的更有效治疗药物提供了基础。
