Van Schaftingen E, Opperdoes F R, Hers H G
Eur J Biochem. 1987 Aug 3;166(3):653-61. doi: 10.1111/j.1432-1033.1987.tb13563.x.
Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.
对布氏锥虫的无细胞提取物进行差速离心后，6-磷酸果糖-2-激酶和果糖-2,6-二磷酸酶表现为胞质酶。这两种活性可以通过在蓝色琼脂糖凝胶和阴离子交换剂上进行色谱分离。6-磷酸果糖-2-激酶对其两种底物的Km值在毫摩尔范围内。其活性依赖于无机磷酸的存在，受磷酸烯醇丙酮酸抑制，但不受柠檬酸或3-磷酸甘油抑制。果糖-2,6-二磷酸酶的Km值为7微摩尔；该酶受1,6-二磷酸果糖（Ki = 10微摩尔）抑制，受6-磷酸果糖、磷酸烯醇丙酮酸和3-磷酸甘油的抑制作用较弱。美拉胂氧对6-磷酸果糖-2-激酶（Ki小于1微摩尔）和果糖-2,6-二磷酸酶（Ki = 2微摩尔）的抑制作用比对丙酮酸激酶（Ki大于100微摩尔）的抑制作用强得多。以葡萄糖作为糖酵解底物时，细胞内2,6-二磷酸果糖和6-磷酸己糖的浓度最高，果糖次之，甘油和二羟基丙酮最低。当与葡萄糖一起添加时，水杨羟肟酸导致2,6-二磷酸果糖、ATP、6-磷酸己糖和1,6-二磷酸果糖的浓度降低。这些研究表明，2,6-二磷酸果糖的浓度主要受6-磷酸果糖-2-激酶底物浓度的控制。磷酸烯醇丙酮酸浓度的变化与2,6-二磷酸果糖对丙酮酸激酶的刺激作用一致。在微摩尔浓度下，美拉胂氧几乎完全阻断了葡萄糖诱导的2,6-二磷酸果糖的形成，而不改变细胞内ATP和6-磷酸己糖的浓度。在较高浓度（3 - 10微摩尔）下，这种药物导致细胞裂解，糖酵解通量成比例下降，以及磷酸烯醇丙酮酸浓度升高，且仅限于细胞外区室。洋地黄皂苷也诱导了类似的变化。得出的结论是，美拉胂氧对布氏锥虫血流形式的裂解作用不是抑制丙酮酸激酶的结果。
