Department of Ecology and Molecular Biology, Section of Microbiology, Royal Veterinary and Agricultural University, Rolighedsvej 21, DK-1958 Frederiksberg C., Denmark.
Appl Environ Microbiol. 1992 Nov;58(11):3638-46. doi: 10.1128/aem.58.11.3638-3646.1992.
Incorporation of [H]leucine and [H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of H and C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 x 10 and 6.6 x 10 cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.
在两个富营养化湖泊中研究了[H]亮氨酸和[H]缬氨酸掺入淡水细菌蛋白质的情况。两种氨基酸的掺入均达到约 50 nM 外部浓度的饱和水平。被吸收的两种氨基酸中只有一部分用于蛋白质合成。在 100 nM 时，细菌分别呼吸了吸收的亮氨酸和缬氨酸的 91%和 78%。亮氨酸的 H 和 C 同位素的呼吸给出了类似的结果。未被呼吸的亮氨酸大部分在细菌蛋白质中回收，而未被呼吸的缬氨酸只有一半左右出现在蛋白质中。在细菌的细胞内池中，[H]亮氨酸在浓度>40 nM 时达到 88%至 100%的同位素饱和。对于[H]缬氨酸，在浓度>80 nM 时获得约 90%的同位素平衡。在典型的 1 小时孵育期内，掺入细菌蛋白质的氚亮氨酸和氚缬氨酸达到 2%至 6%的同位素饱和。在 99 小时批量实验中，根据亮氨酸和缬氨酸的掺入计算的细菌蛋白质合成分别相当于化学测定的蛋白质产生的 31%和 51%（10 nM）和 89%和 97%（100 nM）。测量的 100 nM 亮氨酸和缬氨酸的转换因子分别为 6.4 x 10 和 6.6 x 10 个细胞/mol，并且在预期的理论值范围内。本研究表明，亮氨酸和缬氨酸的掺入都可以真实地测量淡水细菌中的蛋白质合成，并且可以将掺入作为细菌产量的衡量标准。
