COMPARISON OF METHODS FOR EXTRACTION OF BACTERIAL ADENINE-NUCLEOTIDES DETERMINED BY FIREFLY ASSAY
COMPARISON OF METHODS FOR EXTRACTION OF BACTERIAL ADENINE-NUCLEOTIDES DETERMINED BY FIREFLY ASSAY
复制标题
DOI：
10.1128/aem.30.5.713-721.1975
复制
发表时间：
1975-01-01
期刊：
影响因子：
--
通讯作者：
THORE, A
中科院分区：
文献类型：
--
作者：
LUNDIN, A;THORE, A
用10种不同的方法提取大肠杆菌、蜡样芽孢杆菌、肺炎克雷伯菌、金黄色葡萄球菌和铜绿假单胞菌中的腺嘌呤核苷酸。用改进的萤火虫法测定了提取物中的腺苷5′-三磷酸（ATP）、腺苷5′-二磷酸（ADP）和腺苷5′-单磷酸（AMP）。在提取过程中未灭活的细菌酶的分析干扰被认为是一个主要问题。然而，这些酶被灭活在相当大的程度上，包括乙二胺四乙酸在提取试剂。比较了10种提取方法的腺嘌呤核苷酸得率、对酶法测定的干扰、方法的重现性和提取物的稳定性。结果表明，三氯乙酸提取法最能反映完整细菌细胞中ATP、ADP和AMP的实际水平。然而，为了在某些细菌菌株中提取ATP，可以使用几种其他方法，并且从实践的角度来看，这些方法可能是有利的。
Adenine nucleotides inEscherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, andPseudomonas aeruginosawere extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.