SpringerLink - Journal Article

Methods for the measurement of a bacterial enzyme activity in cell lysates and extracts

Brendan P. Burns¹, George L. Mendz² and Stuart L. Hazell¹

(1)	School of Microbiology and Immunology, The University of New South Wales, 2052 Sydney, Australia

(2)	School of Biochemistry and Molecular Genetics, The University of New South Wales, 2052 Sydney, Australia

Abstract The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three different 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 in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity.

Brendan P. Burns
Email: B.Burns@unsw.edu.au

References

1.	Stryer, L. 1988. Biochemistry (3rd Ed). W.H. Freeman & Co: New York.

2.	Jost, P.C., and Griffith O.H. 1982. Lipid-protein interactions. Vol 1. John Wiley & Sons: New York.

3.	Coakley, W., Brown, R.C., and James, C.J. 1973. The inactivation of enzymes by ultrasonic cavitation at 20 kHz. Arch. Biochim. Biophys. 159, 722–729.

4.	Burns, B.P., Hazell S.L., and Mendz, G.L. 1997. In situ properties of aspartate carbamoyltransferase activity in Helicobacter pylori. Arch. Biochem. Biophys. 347, 119–125.

5.	IARC. 1994. Schistosomes, liver fluke and Helicobacter pylori. Abst. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 61, pp. 177–240. Lyon: International Agency for Research on Cancer, World Health Organisation.

6.	Lee, A., Fox, J., and Hazell, S. 1993. Pathogenicity of Helicobacter pylori: a perspective. Infect. Immun. 61, 1601–1610.

7.	Gerhart, J. C., and Pardee, A. B. 1962. The enzymology of control by feedback inhibition. J. Biol. Chem. 237, 891–896.

8.	Mori, M., Ishida, H., and Tatibana, M. 1975. Aggregation and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. Biochemistry 14, 2622–2630.

9.	Else, A. J., and Hervé, G. 1990. A microtitre plate assay for aspartate transcarbamylase. Anal. Biochem. 186, 219–221.

10.	Prescott, L. M., and Jones, M. E. 1969. Modified methods for the determination of carbamoyl aspartate. Anal. Biochem. 32, 408–419.

11.	Allen, C. M., and Jones, M. E. 1964. Decomposition of carbamoyl phosphate in aqueous solutions. Biochemistry 3, 1238–1247.

12.	Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

13.	Lopez, P., and Burgos, J. 1995. Lipoxygenase inactivation by manothermosonication: effects of sonication physical parameters, pH, KCl, sugars, glycerol, and enzyme concentration. J. Agric. Food Chem. 43, 620–625.

14.	Furth, A.J. 1975. Purification and properties of a constitutive β-lactamase from Pseudomonas aeruginosa strain dalgleish. Bioch. Biophys. Acta. 377, 431–443.

15.	Diffley, P. and Jayawardena, A.N. 1982. Comparitive analysis of procedures used to isolate variant antigen from Trypanosoma brucei rhodesien. J. Parasitol. 68, 532–537.

16.	Mendz, G. L., Jimenez, B. M., Hazell, S. L., Gero, A. M., and O’Sullivan, W. J. 1994. De novo synthesis of pyrimidine nucleotides by Helicobacter pylori. J. App. Bacteriol. 77, 1–8.

17.	Mendz, G.L., Hazell, S.L. and Burns, B.P. 1994. Evidence for the Entner-Duodoroff Pathway in Helicobacter pylori. Arch. Biochem. Biophys. 312, 349–356.

18.	Burns, B.P., Hazell, S.L. and Mendz, G.L. 1995. Acetyl Coenzyme A carboxylase Activity in Helicobacter pylori and the requirement of increased carbon dioxide for growth. Microbiology 141, 3113–3118.