Lee W S, Cooper J K, Lynch W H
Can J Microbiol. 1984 Mar;30(3):396-405. doi: 10.1139/m84-058.
Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.
利用来自恒化器培养的荧光假单胞菌的洗涤细胞悬液，检测了由琥珀酸、富马酸和苹果酸形成细胞外产物的酶活性。此前已在荧光假单胞菌中发现了与膜相关的酶活性（葡萄糖、葡萄糖酸和苹果酸脱氢酶），这些酶在碳过量环境中会产生大量细胞外氧化产物。此处开展的研究表明，这种微生物中存在一种苹果酸酶活性，在碳过量环境中可由苹果酸产生细胞外丙酮酸。尽管在碳限制的恒化器培养中，这三种膜脱氢酶的活性显著降低，但发现苹果酸酶活性在这些条件下增加了四倍。苹果酸或琥珀酸对苹果酸脱氢酶和苹果酸酶的调节作用相似。在碳过量培养中，苹果酸脱氢酶活性增加而苹果酸酶活性降低。在碳限制培养中观察到相反的效果。当使用丙酮酸或葡萄糖作为碳源时，苹果酸脱氢酶受可用碳浓度的调节方式相似，但未检测到产生细胞外丙酮酸的苹果酸酶活性。在苹果酸过量培养中产生了大量细胞外草酰乙酸和丙酮酸，但在琥珀酸过量培养中未检测到细胞外氧化产物。这可能是由于在碳过量培养的细胞中，未检测到将添加的外部琥珀酸转化为细胞外富马酸和苹果酸的可检测活性。在碳限制（苹果酸或琥珀酸）培养的细胞中，检测到了将琥珀酸转化为细胞外富马酸和苹果酸的非常活跃的酶。这些碳限制培养的洗涤细胞悬液通过琥珀酸脱氢酶、延胡索酸酶和苹果酸酶的顺序作用，将添加的琥珀酸迅速氧化为细胞外丙酮酸。在以丙酮酸或葡萄糖作为碳源的恒化器培养的细胞中，未检测到产生细胞外产物的琥珀酸脱氢酶和延胡索酸酶活性。还发现，在碳限制（苹果酸或琥珀酸）培养中，琥珀酸、苹果酸和丙酮酸的摄取活性增加，而在碳过量培养中降低。讨论了在碳限制和碳过量环境中，与膜相关的酶形成不同碳异化途径的作用。
