Shieh J, Whitman W B
Department of Microbiology, University of Georgia, Athens 30602.
J Bacteriol. 1988 Jul;170(7):3072-9. doi: 10.1128/jb.170.7.3072-3079.1988.
To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.
为了检测沼泽甲烷球菌细胞提取物中的自养型二氧化碳同化作用，添加乳酸脱氢酶和NADH，将自养合成的乙酰辅酶A形成的丙酮酸转化为乳酸。通过分光光度法测定产生的乳酸。当二氧化碳固定作用朝着乳酸合成的方向进行时，二氧化碳还原为甲烷的过程受到抑制。溴乙烷磺酸盐（BES）是一种有效的产甲烷作用抑制剂，可增强乳酸合成，在没有BES的情况下，甲基辅酶M会抑制乳酸合成。乳酸合成依赖于二氧化碳和氢气，但也观察到了不依赖氢气和二氧化碳的合成。在细胞提取物中，乳酸合成速率约为1.2 nmol·min⁻¹·mg蛋白质⁻¹。添加BES后，乳酸合成速率增加到2.3 nmol·min⁻¹·mg蛋白质⁻¹。由于乙酰辅酶A不会刺激乳酸合成，丙酮酸合酶可能是这些测定中的限制活性物质。¹⁴CO₂中的放射性标记掺入了乳酸中。乳酸C-1、C-2和C-3位置的放射性标记百分比分别为73%、33%和11%。一氧化碳和甲醛均刺激乳酸合成。¹⁴CH₂O特异性掺入乳酸的C-3位，¹⁴CO掺入C-1和C-2位。低浓度的氰化物也会抑制自养生长、一氧化碳脱氢酶活性和自养乳酸合成。这些观察结果与自养型二氧化碳同化的产乙酸途径一致。
