Urease activity, measurement

Urease Activity Measured by Alkatinization of Perfusing Medium... 

Equipment. All potentiometric measurements were taken on a Coming Model 12 research pH meter in conjunction with a Heath-Schlum-berger Model SR-255B strip chart recorder. An Orion Model 95-10 ammonia gas sensing electrode was used for all assays of urease activity. Potentiometric activity measurements were made at 25° in a 10-ml glass thermostatted cell. 

The UBT is based on HP urease activity. The carbon (nonradioac-tive isotope) and " carbon (radioactive isotope) tests require that the patient ingest radiolabeled urea, which is then hydrolyzed by HP (if present in the stomach) to ammonia and radiolabeled bicarbonate. The radiolabeled bicarbonate is absorbed in the blood and excreted in the breath. A mass spectrometer is used to detect carbon, whereas " carbon is measured using a scintillation counter. The stool antigen test is approved by the Food and Drug Administration (FDA), but availability in the United States is limited. It is less expensive and easier to perform than the UBT, and may be useful in children. Although comparable to the UBT in the initial detection of HP, the stool antigen test is less accurate when used to confirm HP eradication posttreatment. Salivary and urine antibody tests are under investigation. ... 

Immobilization of Urease on Sweetzyme Pellets For co-immobilization of urease on the Sweetzyme pellets, 500 ml of 1 g/1 urease solution and 2 g of Sweetzyme pellets was added to a 1-1 beaker [35]. The beaker was left on the benchtop at room temperature for 24 h. The pellets were separated from the solution by decanting and gravity filtration and dried on a paper towel at room temperature for 24 h or until dry. Co-immobilized pellets were stored at 4 °C until use. Activity of immobilized urease was measured at pH 7.5 and 25°C using a standard assay procedure that measures the rate of ammonia liberation [43]. The urease activities obtained with our immobilization procedure were in the range of 550-577 U/g pellets, where a unit liberates 1 pmol of ammonia per minute under the assay conditions. 

Wang and Johnson (2001) reported on test measurement methods that were major indicators of soybean oil quality. These tests included peroxide value, anisidine value, FFA content, phospholipid content, total tocopherol content, oxidative stability index, color, and moisture content. For soybean meal, they reported on urease activity, protein dispersibility index (PDI), rumen bypass or rumen undegradable protein, trypsin inhibitor activity, moisture content, residual oil content, protein content, fiber content, color, amino acid profiles, and protein solubility under alkaline (KOH) conditions. 

The production of ammonia from urea gives rise to a shift of the C(V)-curve along the voltage cLxis as discussed above. The inhibition of the urease activity by Hg " was estimated from the slope of AV versus time plots, compared to the slope without added mercury(II) chloride. In Fig.5, we have shown the observed relative enzyme activity as a function of the Hg -concentration. The detection limit appears to be about 0.001 /xM Hg " " in this measurement system. One sample could be processed within 5 minutes. 

Experiments using C-labeled urea indicated that the microbes that make up the pristine SRPA communities possess considerable potential for urease activity under the correct conditions (Figure 3). Less than 6% of the C-labeled urea was metabolized in each of the treatments shown in Figure 3 indicating that the urea available to the cells for hydrolysis was probably not depleted in such a way that the rates would be altered. The highest rate of urea hydrolysis (> 800 finol urea hydrolyzed/mL/h) was noted for SRPA consortia that were supplemented with 0.01% molasses. The same consortia provided with less molasses (0.001% and 0.00075%) still showed marked urea hydrolysis (ca. 400 finol urea hydrolyzed/mL/h). These values are higher than those measured for B. pasteurii... 

Urease activity was determined from the decomposition of rrrea irrto arrtmorria arrd carbon dioxide CO with a pH-sensitive dye Bromocresol Violet The reaction rrrixture corrtained a necessary amormt of urea and 0.015 rtiM Bromocresol Violet adjrrsted to pH 6.2. The known ntrmber of PEMC containing ttrease was added to the solutioa Then, the kinetics of the reaction was measured from a change in absorption of the dye at 588 run [15]. The activity of the free enzyme in the preserrce of the polyelectrolyte was measured by a similar method, a necessary amourrt of the polyelectrolyte with pH adjusted to 6.2 was added to the reaction solution. 

The situation is quite different when urease activity is measured in the intact organism. There is little measured activity until pH 6.5 is reached, and then there is a greater than 20-fold increase in urease activity, with a steady state of urease activity being held down to a pH of 2.5. There is, therefore, medium acidity activation of urease activity in the intact bacteria. The maintained intrabacterial urease activity in acid is a major factor in the acid adaptation of H. pylori. It is predicted, then, that intrabacterial urease activity will be able to buffer both periplasm and cytoplasm in gastric acid. These measurements are made by determining the amount of released from urea in buffer. 

Most previous measurements of H. pylori urease activity have been done using the rate of change of pH of an unbuffered medium as pH increases, urease activity is lost, hence, blinding the investigator to the acid activation of intrabacterial urease. 

The rate of alkalinization is measured in a chamber containing H. pylori after superfusion at the different pH values indicated at the top of the figure and stopping the superfusion using a pH sensor. There is no alkalinization in a weakly buffered solution containing glucose, but when 2.5 mM urea is present in the medium, there is an increase of pH at all the pH values. The increase is limited until pH 3.0, at which there is a 40-fold increase in alkalinization due to intrabacterial urease activity an increase is still seen at pH 2.5. The large increase in urease activity seen at pH 3.0 is due to the inability of the urease activity to restore periplasmic pH so as to shut down the acid-activated urea channel, Urel. 

A similar activation of urea hydrolysis is seen when pH measurements are made in the cytosensor and the rate of alkalinization correlated with urease activity, as shown in the figure on the opposite page. This also differs from previous investigations, because acid is constantly added to the organisms, and inactivation of the mechanism for enhancement of urease activity does not occur. There is approximately a 40-fold activation of urease activity as the pH falls to below 4.0. At higher pH, in the small volume of the chamber, urease activity is able to elevate chamber pH very rapidly hence, urease activity is only transient. At a pH of 3.0, the level of acid is sufficient to require significant levels of urease activity to attempt to elevate pH. These... 

Here urease activity present in the intact organism was measured, and urease activity that was removed by washing the surface was measured as a function of fixed medium pH using " C02 liberated from labeled urea. 

When experiments are carried out to measure the effect of urease activation on periplasmic pH or transmembrane potential, it is found that the addition of urea at pH 3, 4 or 5 brings periplasmic pH to a value of 6.2 and the transmembrane potential to a value of 105 mV. The driving force for entry of H across the ATP synthase is therefore maintained constant at about -220 mV... 

The relative reaction rates of urease at low water activities have been measured (105). Mixtures of lyophilized urease and 14C-urea were exposed to various relative humidities at 20°. The rates observed ranged from negligible activity below 60% humidity to full activity at 100% relative humidity. It was estimated that hydrolysis could occur only after the water content exceeded one molecule per polar group. 

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