Atkinson W H, Winkler H H
Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile 36688.
J Bacteriol. 1989 Feb;171(2):761-6. doi: 10.1128/jb.171.2.761-766.1989.
Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation.
普氏立克次氏体可从[腺嘌呤 - 2,8 - 3H]NAD积累放射性，但不能从[烟酰胺 - 4 - 3H]NAD积累，这表明NAD并非完整摄取。细胞外NAD被立克次氏体水解，水解产物烟酰胺单核苷酸和AMP以时间和温度依赖的方式出现在孵育培养基中。颗粒（膜）部分含有90%的这种NAD焦磷酸酶活性。用[腺嘌呤 - 2,8 - 3H]NAD孵育后积累了放射性标记的立克次氏体被提取出来，其细胞内成分通过色谱法进行分析。细胞含有标记的AMP、ADP、ATP和NAD。NAD衍生的细胞内AMP通过一条与先前描述的AMP转位酶不同且额外的途径运输。外源性AMP（1 mM）抑制了[腺嘌呤 - 2,8 - 3H]NAD的放射性摄取以及细胞外NAD的水解。AMP增加了细胞内以NAD形式存在的放射性标记的百分比。烟酰胺单核苷酸不被立克次氏体摄取，不抑制细胞外NAD的水解，也不是[腺嘌呤 - 2,8 - 3H]NAD放射性摄取的良好抑制剂。AMP和ATP（两者均可运输）都不能支持细胞内NAD的合成。在一种不能运输完整NAD的生物体中存在细胞内[腺嘌呤 - 2,8 - 3H]NAD，这表明在NAD水解和转位位点由AMP重新合成了[腺嘌呤 - 2,8 - 3H]NAD。
