MethodsfortheMeasurementofaBacterialEnzymeActivityinCellLysates

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

Studies of bacterial enzyme activities provide fundamental information relevant to microbial physiology, and to a more complete understanding of cell metabolism, bacterial evolution, and to the interactions that occurs between bacterium and host. Characterizing enzyme activities helps to elucidate regulatory mechanisms and pathways in an organism, and to identify proteins essential for cell survival. The ability to investigate enzyme activities in whole cell lysates or crude extracts is important for the initial identification of a particular activity, and for obtaining information on enzyme function in an environment close to the cellular milieu.

The activities of enzymes in the intracellular milieu depend on their intrinsic physico-chemical properties and on their interactions with other cellular components. Modulation of enzyme activities of functional relevance occurs not only among the constituents of recognised multienzyme clusters, but also among soluble enzymes and other cellular components (1, 2). Proteins have also been known to be more stable in concentrate than in dilute solutions (3). The basic mechanisms of action of soluble enzymes established in purified preparations are not expected to change from the in vivo conditions, however their activities are modulated by the intracellular milieu. Thus, in situ investigations of enzyme activities may reveal properties which would otherwise remain undetected.

The investigation of enzyme activity with a variety of methods results in synergies in the understanding of the characteristics of an enzyme. Application of different techniques can validate data obtained by one method by the results of the others, yield more specific information on a particular aspect of a system, and provide results in a range of assay conditions ordinarily not available to a single method. The benefits and disadvantages of particular techniques, such as sensitivity, efficiency, and cost, need to be considered to determine which are the most useful methods for a specific situation.

In a previous investigation (4), we used three different techniques to study aspartate carbamoyltransferase (ACTase) activity in Helicobacter pylori. This bacterium is an important human pathogen (5, 6), and the enzyme of interest is a key regulatory step in bacterial de novo pyrimidine nucleotide metabolism (7). As this enzyme appears to be essential for the survival of the bacterium, it provides a potential site for therapeutic intervention. Consequently, an in-depth understanding of the enzyme activity and regulation in situ would serve toward more rational design of therapeutic agents.

Preparation of cell free extracts

Cells were grown at 37°C in an atmosphere of 10% CO2 in air, and 95% humidity. Bacteria were harvested during the late logarithmic growth phase from agar plates into 0.1 M Tris buffer (pH 8.0). H. pylori cells change shape as cultures grow older. The bacteria have spiral-rod forms when they are in log phase, and this shape becomes spherical with the formation of coccoids which tend to aggregate in the stationary phase. Cultures on plates will produce cells of different ages, but based on the morphology of the bacteria, phase contrast microscopy allows easy determination of the predominant growth phase. Under our experimental conditions, inspection of cultures showed that at 24h growth more than 95% of the cells were in the spiral-rod form, and after 72h growth more than 95% of the cells were coccoids. It should be noted that the formation of coccoid aggregates poses a problem for the estimation of cell growth by spectrophotometry at 600 nm. The number of scattering centres can decrease significantly upon aggregation, and the measurements could underestimate bacterial growth.

Harvested cells suspended in Tris buffer were centrifuged at 17,000g for 8 min at 4°C. The resulting pellet was washed twice and resuspended in the same buffer. Cells were lysed by twice freezing in liquid nitrogen and thawing the suspensions, which were allowed to thaw completely before re-freezing. To obtain crude extracts, the lysates were centrifuged at 27,000g for 8 min at 4°C, and the supernatant carefully separated from the pellet made up of cell-envelope debris. The lysates and the crude extracts could be stored at -20°C for several months without loss of activity.

Measurement of ACTase activity

Nuclear magnetic resonance spect roscopy (NMR)

The unique potential of NMR spectroscopy for monitoring simultaneously the concentrations of several metabolites in complex milieux makes it one of the most powerful techniques available to carry out this type of study.

For NMR measurements, lysates or cell-free extracts were resuspended in 0.15 M NaCl constituted in 5:1 H2O/²H2O buffer mixtures to provide deuterium frequency lock for the spectrometer. Substrate concentrations were 40 mM L-aspartate and 50 mM carbamoyl phosphate, in 0.1 M HEPES buffer (pH 8.0). The reaction was started by adding 100 μl of cell free extract to a total sample volume of 600 μl. To allow for efficient dispensing of the assay mixture into the 5 mm narrow bore NMR tube (Wilmad, Buena, NJ), the lysate or extract suspension and the substrates were mixed in an Eppendorf tube; diluting viscous cell lysates helped to place them into the tube.

Free induction decays were collected using a Bruker AM-500 spectrometer, operating in the Fourier transformation mode. Measurements were carried out at 37°C, and sequential spectra were acquired automatically at 500.11 MHz with presaturation of the water resonance. The instrumental parameters were: spectral width 5347 Hz, memory size 8 K, recycling time 3.5 s, number of transients 144, and pulse angle 50° (8 μs). To improve signal-to-noise, exponential filtering of 1 Hz was applied prior to Fourier transformation. Depending on the viscosity of the sample and the concentrations of the substrates, it is often advisable to employ resolution-enhancing window functions at the expense of losing some signal intensity; for example, a Gaussian window function with typical parameters: -1 Hz line broadening, and 0.19 Gaussian parameter.

The time evolution of the utilisation of substrates and appearance of product was followed by acquiring sequential spectra of the reactions. Progress curves were obtained by measuring the integrals of substrate and product resonances at each point in time. Maximal rates were calculated from good fits (correlation coefficients ³ 0.99) of the data to straight lines for 30 min of the reactions.