A continuous assay for an intracellular enzyme: The analysis of acetate kinase in Escherichia coli
✍ Scribed by H.Michael Koplove; Charles L. Cooney
- Publisher
- Elsevier Science
- Year
- 1976
- Tongue
- English
- Weight
- 452 KB
- Volume
- 72
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A continuous, automated assay for the measurement of acetate kinase activity in Escherichia co/i is described. The assay system consists of three steps: continuous sonication, acetate kinase catalyzed production of ATP, and subsequent measurement of ATP concentration. Complete extraction of acetate kinase was obtained with sonicator residence times greater than 1.5 min and minimal enzymatic deactivation was observed with residence times shorter than 4 min. The acetate kinase catalyzed reaction, which is nonisothermal, yielded linear calibration curves when tested with purified acetate kinase and E. co/i whole broths containing acetate kinase. The overall system responds with an I I-min transport delay in the Autoanalyzer and exhibits a 2-min first-order response. The approach taken in the development of this analysis is applicable to the rapid analysis of other intracellular products.
Acetate kinase (ATP: acetate phosphotransferase; EC 2.7.2.1) is an intracellular enzyme which functions in ATP synthesis and/or acetate utilization in facultative and anaerobic microorganisms (1,2). When coupled with adenylate kinase (ATP: AMP phosphotransferase; EC 2.7.4.3), acetate kinase has been effective for the in vitro regeneration of ATP from AMP (3). In a recent investigation, having the objective of increasing the microbial productivity of acetate kinase for use in such an ATP regeneration system, Koplove and Cooney (in preparation) observed transiently higher enzyme activities following nutritional "shiftups" in chemostat cultures. Studying the time course of transient enzyme activities required frequent sampling. The standard noncontinuous assay, which consisted of enzyme extraction, enzyme-catalyzed reaction. and subsequent measurement of the reaction velocity, was unsatisfactory for analyzing the large number of samples necessary. Even though no purification of acetate kinase from cell extracts was required, the assay took several hours to complete, during which time proteolytic activity, oxidation, and other types of deactivation could diminish the apparent enzyme activity. Furthermore, by not having a rapid enzyme assay to feed back information to the investigators, it was difficult to determine whether the samples were being taken during the critical time span in the experiment. For these reasons, a continuous and rapid procedure for analyzing this 297