Li Y, Gray G R
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1996 Dec 17;35(50):16299-304. doi: 10.1021/bi961820k.
Lipids extracted from Bacillus subtilis using a 2:1 mixture of chloroform and methanol have been found to be very mitogenic. These lipids were fractionated on a silica column and eluted with chloroform, acetone, and 60% methanol in chloroform, and the mitogenic activity was recovered in the last fraction. Further purification of the mitogenic components was achieved by HPLC on an amino-isopropyl bonded-phase column using a linear gradient of 5-20 mM ammonium acetate in a mobile phase consisting of hexane, 2-propanol, methanol, and water (5.5:8:1.5:1). Two major and several minor mitogenic peaks were observed. One major mitogenic lipid was isolated in pure form and structurally characterized by chemical degradation analysis, NMR spectroscopy, and mass spectrometry. Mild acid hydrolysis of the lipid released glycerol phosphate and a neutral glycolipid. Saponification of the lipid released a water-soluble head group and C14-C17 branched fatty acids. Total acid hydrolysis of the head group revealed the presence of glycerol and glucose in a ratio of 1:1. Mild acid hydrolysis of the head group to remove the glycerol phosphate produced a neutral partial head group. The partial head group was methylated and then analyzed by GLC-CIMS and by the reductive-cleavage method, which revealed that it was composed of nonreducing terminal glucopyranosyl, 6-linked glucopyranosyl, and 3-linked glycerol residues in equimolar proportions. Finally, the molecular weight of the permethylated head group, obtained by fast atom bombardment mass spectrometry, was 724.3340, which is consistent with the composition of two glucose residues, one glycerol residue, and one glycerol phosphate residue. On the basis of all these results, the intact mitogenic lipid was identified as 1,2-di-O-acyl-3-O-[6-(sn-glycerol-phospho)-beta-D-glucopyranosyl-(1-->6) - beta-D-glucopyranosyl]glycerol. The purified glycolipid possessed very potent mitogenic activity in a murine splenocyte proliferation assay at a concentration of 0.01-0.1 microgram/mL.
已发现，用氯仿和甲醇的2:1混合物从枯草芽孢杆菌中提取的脂质具有很强的促有丝分裂活性。这些脂质在硅胶柱上进行分馏，并用氯仿、丙酮和氯仿中的60%甲醇洗脱，促有丝分裂活性在最后一个馏分中得以恢复。通过在氨基异丙基键合相柱上进行高效液相色谱法（HPLC）进一步纯化促有丝分裂成分，流动相为己烷、2-丙醇、甲醇和水（5.5:8:1.5:1）的混合液，其中含有5 - 20 mM醋酸铵的线性梯度。观察到两个主要的和几个次要的促有丝分裂峰。以纯形式分离出一种主要的促有丝分裂脂质，并通过化学降解分析、核磁共振光谱法和质谱法对其结构进行了表征。该脂质经温和酸水解后释放出甘油磷酸和一种中性糖脂。脂质皂化后释放出一个水溶性头部基团和C14 - C17支链脂肪酸。头部基团的总酸水解显示甘油和葡萄糖的比例为1:1。对头部基团进行温和酸水解以去除甘油磷酸，得到一个中性部分头部基团。该部分头部基团经甲基化后，再通过气相色谱 - 化学电离质谱法（GLC - CIMS）和还原裂解法进行分析，结果表明它由等摩尔比例的非还原性末端吡喃葡萄糖基、6 - 连接的吡喃葡萄糖基和3 - 连接的甘油残基组成。最后，通过快原子轰击质谱法测得全甲基化头部基团的分子量为724.3340，这与两个葡萄糖残基、一个甘油残基和一个甘油磷酸残基的组成相符。基于所有这些结果，完整的促有丝分裂脂质被鉴定为1,2 - 二 - O - 酰基 - 3 - O - [6 - (sn - 甘油 - 磷酸) - β - D - 吡喃葡萄糖基 - (1→6) - β - D - 吡喃葡萄糖基]甘油。在小鼠脾细胞增殖试验中，纯化的糖脂在浓度为0.01 - 0.1微克/毫升时具有非常强的促有丝分裂活性。
