J

Figure 9.89 Pyrimidine 5'-nucleotidase assay in undialyzed human erythrocyte lysate obtained by 1:5 dilution of packed cells with deionized water, (a) Separation of 1 nmol of CMP, cytidine, and uridine as the standards. Separation of the assay mixture, containing 50 mM Tris-HCl, pH 7.5, 0.2 mM CMP, 1 mM MgCl2, 1 mM DTT, and 20 fiL of lysate in 0.5 mL of total volume (b) at time zero and (c) after 30 minutes of incubation (c). (From Amici et al., 1994.)

phosphate as the substrate, using a continuous spectrophotometric method as described in Chapter 1 (Section 1.3.1). More recently, the HPLC method has been used together with a nucleoside monophosphate such as AMP as the substrate. In an assay of Rossomando et al. (1983), the formation of adenosine during the course of the reaction was monitored at 254 nm.

Substrate and product were separated by reversed-phase HPLC (/xBonda-pak) using a mobile phase of a phosphate buffer at pH 5.5 with 1% methanol. The column was eluted isocratically, and the detection was at 254 nm.

The reaction mixture contained the substrate and buffer, and the reaction was started by the addition of the enzyme. In one study, the substrate was formycin 5'-monophosphate, a fluorescent analog of AMP. The formation of formycin A, the analog of adenosine, is shown in Figure 9.90 as a function of incubation time.

In an interesting application of this assay to the question of reaction mechanisms, the substrate AMP was replaced by the thioanalog 5'-deoxy-5'-thioadenosine monophosphate [A(S)MP]. The structure of A(S)MP is shown in Figure 9.91. (This analog is available from Calbiochem-Behring.) With this analog it was possible to explore the question of whether the enzyme cleaved between the C-5' and the oxygen atoms or between the oxygen atom and the

12 10

FoMP

Figure 9.90 HPLC analysis of a reaction mixture containing AMP and alkaline phosphatase. Tracings obtained of reaction mixture (A) immediately after the addition of enzyme, (B) after 10 minutes, and (C) after 15 minutes. (From Rossomando et al., 1981a.)

phosphorus atom. These alternatives are illustrated in Figure 9.92. It is clear that the site of cleavage can be distinguished, since the alternatives will produce different reaction products: in one case thioadenosine and phosphate, in the other, thiophosphate and adenosine.

Since thioadenosine is readily separated from adenosine by HPLC, the use of this analog together with the HPLC assay method allowed the site of cleavage to be established. As shown in Figure 9.93/4, an analysis of the incubation mixture during the course of the reaction revealed the formation of thioadenosine. No adenosine was detected. These findings supported the conclusion that the site of cleavage was the bond between the sulfur and the phosphate.

This analog proved to be useful in another way as well when it was found that the analog was not a substrate for a 5'-nucleotidase, since, as shown in Figure 9.93B, incubation of the thio analog with this activity produced no

OH OH

OH OH

5'-OEOXY-5'-THIOAOENOSINE S'-MONOPHOSPHATE [A(S)MP]

0 II

OH OH

5'-0E0XY-5'-THI0IN0SINE 5'-MONOPHOSPHATE [l(S)MPj

Figure 9.91 Structure of the thio analogs of AMP and IMP in which sulfur replaces the bridge oxygen between the 5' carbon and the phosphorus. (From Rossomando et al., 1983.)

BASE

0 II

PME PME

Figure 9.92 Sites of bond cleavage by phosphomonoesterases. Arrow 1 indicates cleavage of the bond between the phosphorus and sulfur atoms. The products are shown in reaction (1). Arrow 2 indicates cleavage between the carbon and the sulfur, and the reaction products are shown in reaction (2).

0.04

2 ooo bJ

0.00

0 5 10 19 20 25 RETENTION TIME (min)

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (A) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 (jlL containing 100 mAi Tris-HCl (pH 8.1), 300 (iM A(S)MP, and 20 mAi MgCl2, the reaction was initiated by addition of 2 /ug of enzyme and incubated at 30°C for 6 hours. A 20 fiL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom 5'-nucleotidase incubated with A(S)MP. In a reaction volume of 100 (iL containing 100 mAi Tris-Cl (pH 8.1), 300 fiM A(S)MP, and 20 mAi MgCl2, the reaction was initiated by addition of 6 /xg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 /xL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)

reaction product. These results suggest the possibility that this thio analog and the HPLC assay method may be useful for discriminating between the 5'-nucleotidase and alkaline phosphatase activities.

As mentioned, the foregoing activities can also be assayed using phenylpho-sphate as substrate. In the assay described by Togari et al. (1987), electrochemical detection provided high sensitivity.

In this assay, the product, phenol, was separated by chromatography on a Develosil ODS-7 analytical column (4.6 mm X 150 mm). The mobile phase was a 70:30 mixture of 10 mM acetate buffer (pH 4.0) and methanol at a flow rate of 1.5 mL/min. For detection of phenol, the electrode potential was set at 1.2 V against an Ag/AgCl reference electrode.

The standard incubation mixture for assay of alkaline phosphatase, contained in a total 200 ¡iL\ 5 mAi disodium phenylphosphate, 50 mAi carbonate buffer (pH 10.2), saliva, and distilled water. The reaction was started by addition of saliva and was carried out at 37°C for 30 minutes. The reaction was

1 -

1

t i

-A(S)MP

B -

(S)A4o ~ 1 ■

' i

i i

0 5 10 19 20 25 RETENTION TIME (min)

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (A) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 (jlL containing 100 mAi Tris-HCl (pH 8.1), 300 (iM A(S)MP, and 20 mAi MgCl2, the reaction was initiated by addition of 2 /ug of enzyme and incubated at 30°C for 6 hours. A 20 fiL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom 5'-nucleotidase incubated with A(S)MP. In a reaction volume of 100 (iL containing 100 mAi Tris-Cl (pH 8.1), 300 fiM A(S)MP, and 20 mAi MgCl2, the reaction was initiated by addition of 6 /xg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 /xL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)

terminated by adding 50 /xL of 25% trichloroacetic acid. After centrifugation, a 20 /xL aliquot of the supernate was analyzed by HPLC. The assay of acid phosphatase was carried out in the same manner except that 50 mM citrate (pH 4.8) was used as the buffer. The formation of phenol was linear for up to 60 minutes and 40 fiL of saliva as enzyme source.

Whole saliva and duct saliva from parotid and sublingual glands were used as the sources of the acid and alkaline phosphatases studied.

9.9.4 Adenosine Deaminase (Ubertl et al., 1977; Hartwick et al., 1978)

Adenosine deaminase catalyzes the deamination of adenosine to form inosine and ammonia. The inosine (Ino) can be degraded further to hypoxanthine (Hyp) by nucleoside phosphorylase, an activity often present in extracts. Therefore, in many cases, the assay involves a determination of either the loss of adenosine (Ado) or the formation of both inosine and hypoxanthine. An early study by Uberti et al., 1977, was followed by another by Hartwick et al., 1978.

In the study by (Hartwick et al., 1978), the compounds were separated by reversed-phase HPLC using columns prepacked with Qg /xBondapak or Par-tisil ODS. Compounds were eluted isocratically using a mobile phase of methanol and 10 mM KH2P04 (14:86, v/v) with no further pH adjustment. The separations obtained using these conditions are shown in Figure 9.94/4.

The activity was obtained from a lysate of red blood cells. The reactions were terminated with a boiling water bath (45 s), and the samples clarified by centrifugation. Samples of 5 /xL were analyzed. Figure 9.94B, C, D shows the chromatographic profiles obtained after incubation times of 3,30, and 50 minutes, respectively, with the enzyme. The loss of adenosine is noted, but the effect of the nucleoside phosphorylase is seen, since hypoxanthine, not inosine, is the final product.

9.9.5 AMP Deaminase (Jahngen and Rossomando, 1984; Raffln and Thebauit, 1991)

The enzyme adenylic acid deaminase catalyzes the deamination of AMP to IMP and ammonia. For the HPLC method, the assay involves the separation of the substrate, AMP, from the reaction product IMP. The enzyme is found in muscle.

In the assay of Jahngen and Rossomando (1984), AMP and IMP were separated by means of ion-paired reverse-phased HPLC on a Ci8 (/xBondapak) column with a mobile phase of 65 mM potassium phosphate (pH 3.6), 1 mM tetra-n-butylammonium phosphate, and 4% methanol. The column was eluted isocratically, and the eluent was monitored at 254 nm. When the formycin analogs were used, detection was at 295 nm; at this wavelength there was no

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