Goelz S E, Cronan J E
Biochemistry. 1982 Jan 5;21(1):189-95. doi: 10.1021/bi00530a032.
Previous genetic and biochemical experiments have suggested that the adenylate kinase of Escherichia coli may be directly involved in phospholipid synthesis through formation of a complex with sn-glycerol-3-phosphate acyltransferase, the membrane-bound enzyme that catalyzes the first step in phospholipid synthesis. In this paper we report direct experiments to test this hypothesis. A mutation within the adenylate kinase structural gene is described that results in a temperature-sensitive phospholipid synthesis (assayed in vivo) and a temperature-sensitive acyltransferase. The adenylate kinase activity of this strain is only minimally altered either in vitro or [as assayed by adenosine 5'-triphosphate (ATP) levels] in vivo. This result demonstrates that the inhibition of phospholipid synthesis is not the result of reduced ATP levels. We report the purification of E. coli adenylate kinase to homogeneity; and find that the addition of homogeneous wild-type adenylate kinase to membranes containing a mutationally altered temperature-sensitive acyltransferase results in thermal stabilization of the acyltransferase activity. Ovalbumin has no such protective effect. Purified E. coli inner membranes contain several proteins that are precipitated by addition of anti adenylate kinase antibody to detergent-solubilized membranes.
先前的遗传学和生物化学实验表明,大肠杆菌的腺苷酸激酶可能通过与sn-甘油-3-磷酸酰基转移酶形成复合物,直接参与磷脂合成。sn-甘油-3-磷酸酰基转移酶是一种膜结合酶,催化磷脂合成的第一步。在本文中,我们报告了直接实验来检验这一假设。描述了腺苷酸激酶结构基因内的一个突变,该突变导致温度敏感的磷脂合成(体内测定)和温度敏感的酰基转移酶。该菌株的腺苷酸激酶活性在体外或体内(通过三磷酸腺苷(ATP)水平测定)仅有微小改变。这一结果表明,磷脂合成的抑制不是ATP水平降低的结果。我们报告了将大肠杆菌腺苷酸激酶纯化至同质;并且发现,向含有经突变改变的温度敏感酰基转移酶的膜中添加同质野生型腺苷酸激酶会导致酰基转移酶活性的热稳定。卵清蛋白没有这种保护作用。纯化的大肠杆菌内膜含有几种蛋白质,通过向去污剂溶解的膜中添加抗腺苷酸激酶抗体可使其沉淀。