Quantitative IR studies of acyl chain conformational order in fatty acid homogeneous membranes of live cells of Acholeplasma laidlawii B.
Quantitative IR studies of acyl chain conformational order in fatty acid homogeneous membranes of live cells of Acholeplasma laidlawii B.
复制标题
无胆原体活细胞脂肪酸均质膜中酰基链构象顺序的定量红外研究。
DOI：
10.1021/bi00075a023
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Mendelsohn,R
中科院分区：
文献类型：
--
作者：
Moore,DJ;Wyrwa,M;Reboulleau,CP;Mendelsohn,R
材料和方法细胞生长。无胆原体莱氏 B 细胞在 37°C 的去脂培养基（pH 8.3）中生长，该培养基由以下成分组成：Bacto 心脏浸液肉汤 (12 g/L)、Bacto 蛋白胨 (5 g/L)、Bacto 酵母提取物 (5 g/L)、葡萄糖 (2.5 g/L)、Tris (3.5 g/L)、牛血清白蛋白 (4 g/L) 和青霉素 G （105 单位/升）。添加抗生物素蛋白以抑制脂肪酸从头合成（Silvius & McElhaney，1978）。添加十五烷酸、肉豆蔻酸或棕榈酸作为浓乙醇溶液，得到 120 µ 的最终浓度。通过离心收获细胞；浓缩细胞悬液用于红外实验。将活细胞沉淀放置在两个 Cap2 窗口之间，并用 12 µ Teflon 垫片隔开。在某些情况下，通过在 39°C 结束红外实验、将细胞放回琼脂平板并在 37°C 重新培养细胞来确认活细胞的活力。大多数使用活细胞沉淀的红外实验都进行到 70°C，在该温度下细胞不再存活。图1a将在39°C下的富含肉豆蔻酸的活细胞沉淀物的对称CH2伸缩频率的热致响应与在70°C下来自同一细胞培养物的第二个沉淀物的对称CH2伸缩频率的热致响应重叠。图1b和图1c分别是富含十五烷酸和棕榈酸的活细胞的等效数据。这些结果在三个系统中的重现性以及可行性研究提供了令人信服的证据，证明在低于约 40°C 的温度下（即使在那些温度为 70°C 的实验中），我们的光谱来自活细胞。
MATERIALS AND METHODSCell Growth. Acholeplasma laidlawii B cells were grown at 37 C in a lipid-depleted medium, pH 8.3, consisting of the following: Bacto heart infusion broth (12 g/L), Bacto peptone (5 g/L), Bacto yeast extract (5 g/L), glucose (2.5 g/L), Tris (3.5 g/L), bovine serum albumin (4 g/L), and penicillin G (105 units/L). Avidin was added to inhibit de novo fatty acid synthesis (Silvius & McElhaney, 1978). Pentadecanoic, myristic, or palmitic acids were added as concentrated ethanolic solutions to give a final concentration of 120 µ. Cells were harvested bycentrifugation; concentrated cell suspensions were used for IR experiments. The pellet of live cells was placed between two Cap2 windows separated with a 12-µ Teflon spacer. On several occasions, the viability of the live cells was confirmed by ending an IR experiment at 39 C, returning the cells to agar plates, and reculturing the cells at 37 C. Most IR experiments with live cell pellets were taken to 70 C, at which temperature the cells are no longer viable. Figure la overlays the thermotropic response of the symmetric CH2 stretching frequency of a myristic acid enriched live cell pellet taken to 39 C with that of a second pellet, from the same cell culture, taken to70 C. Figure lb and Figure lc are the equivalent data for pentadecanoic acid and palmitic acid enriched live cells, respectively. The reproducibility of these results within each of the three systems along with the viability studies provide compelling evidence that at temperatures below about 40 C (even in those experiments taken to 70 C) our spectra are derived from live cells.