MethodsfortheMeasurementofaBacterialEnzymeActivityinCellLysates4

The use of a micro-titre based colorimetric assay provided a third method for the study of ACTase activity, and the most useful for large scale measurements. The method was essentially as described previously (9), but several findings proved useful in optimising the assay for this work. Although the authors suggest that the colour development reagents can be stored for a short period of time (10), it was found in the present study that the most consistent results were obtained when the reagents were made fresh immediately prior to use. In constructing the CAA standard curve in Figure 2, it was found that the absorbance values increased linearly with respect to CAA up to 0.15 μmole CAA, slightly more than the 0.125 μmole reported previously. At CAA quantities higher than this, the readings suffered from noise and were no longer linear with respect to CAA. This was important in assessing that this technique could not be used to study the effects of CAA on ACTase activity, as described above. For CAA quantities below 0.015 μmole, the readings were below the limits of sensitivity of the assay. Comparison of colour development times between 2 and 7 h, revealed that a two hour development time yielded the most consistent and reproducible results.

The colorimetric assay was adopted as the preferred method, because its protocol was the more efficient and inexpensive. Employing this assay, several 96-well plates could be used, allowing for the processing of hundreds of samples simultaneously. Experiments could be performed routinely in at least triplicate, increasing their precision. The colorimetric technique provided significant efficiency improvements compared with the processing time of both NMR and the radioactive analysis methods. A novel adaptation of this technique was also seen with the use of this method to detect ACTase activity in a native PAGE gel. A single area of ACTase activity was localised approximately 8 mm down on a 6% alcrylamide gel (Figure 3), suggesting the presence of a high molecular weight protein. This modification of this microtitre assay for use as an enzyme activity stain may be prove useful particularly for protein isolation and purification.

The ability to include a good number of controls in the measurements also ensured very reliable measurements of ACTase activity. No significant increase in optical density was observed between controls with only enzyme extracts and samples with enzyme extracts together with one of the substrates, suggesting there was little or no formation of ureido products other than carbamoyl aspartate. No increase in optical density was observed when carbamoyl phosphate and aspartate were incubated without cell-free extracts, suggesting that any chemical formation of carbamoyl aspartate was below the detection limits of the method. Table 2 is a summary of the results on ACTase activity using this method, showing the significant data generated using this technique.

The main limitation found with the colorimetric assay was already described, namely the inability to study the effect of the end-product CAA on enzyme activity. Lack of results in detecting ACTase activity in whole cells was thought to be another limitation of the method. However, the finding that [¹⁴C]carbamoyl phosphate was not transported into the cells, indicated that the lack of detection of enzyme activity in whole cells was a feature of the permeability of the H. pylori cell membrane, and not a deficiency of the colorimetric assay.

In conclusion, this study has demonstrated the benefits of using several enzyme assays to investigate in situ the functioning of the ACTase enzyme in H. pylori. The use of a direct NMR method, a sensitive radioactive assay, and a very efficient micro-titre assay allowed for a comprehensive understanding of this activity in a complex background. Further use of such a combination of methods should allow for more complete understandings of bacterial enzyme systems in situ.