Blum J J
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Eukaryot Microbiol. 1996 Mar-Apr;43(2):144-50. doi: 10.1111/j.1550-7408.1996.tb04495.x.
Leishmania major promastigotes were treated with digitonin and the rates at which [1-14C]acetate, [1,4-14C]succinate, [1-14C]glutamate, and [U-14C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl-CoA synthase. Its rate of oxidation was negligible if the permeabilized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine. Ca++ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD-to-cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca++, hyperosmolality, and Krebs cycle intermediates.
用洋地黄皂苷处理硕大利什曼原虫前鞭毛体，并在合适的辅因子存在的情况下，测定[1-¹⁴C]乙酸盐、[1,4-¹⁴C]琥珀酸盐、[1-¹⁴C]谷氨酸盐和[U-¹⁴C]丙氨酸的氧化速率。在所检测的四种底物中，乙酸盐的氧化速率最低，即使添加了烟酰胺腺嘌呤二核苷酸（NAD）、辅酶A（CoA）、二磷酸腺苷（ADP）和乙酰辅酶A合酶也是如此。如果在添加辅因子之前洗涤通透细胞，其氧化速率可忽略不计，这表明需要一种未知因子。琥珀酸盐的氧化速率比完整细胞氧化它的非常缓慢的速率要高得多。其氧化速率受到抗霉素A的强烈抑制，但谷氨酸盐的氧化速率几乎不受影响。延胡索酸盐抑制乙酸盐、谷氨酸盐和琥珀酸盐的氧化速率，但增加丙氨酸的氧化速率。钙离子（Ca²⁺）抑制丙氨酸和琥珀酸盐的氧化速率，但不抑制乙酸盐或谷氨酸盐的氧化速率。通过添加甘露醇增加渗透压部分抑制丙氨酸的氧化速率，但对谷氨酸盐的氧化速率影响很小。这些结果表明，通透细胞中仍存在可观的转氨酶活性，并支持早期数据表明存在一个分支的烟酰胺腺嘌呤二核苷酸到细胞色素氧化酶系统。这些结果还提供了关于这两个分支对钙离子、高渗和三羧酸循环中间产物敏感性的初步信息。
