Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts

• Main Authors: Burns Brendan, Mendz George, Hazell Stuart
• Format: Article
• Language: English
• Published: BMC 1998-01-01
• Series: Biological Procedures Online
• Subjects: enzymes; bacteria; research design
• Online Access: http://www.biologicalprocedures.com/bpo/arts/1/5/m5.htm

<p>The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of <it>Helicobacter pylori</it> by three diffirent methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity <it>in situ</it>. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity.