Analysis by Thin Layer Chromatography (TLC) of nucleotides released during

It was of interest which nucleotides were produced during incubation of the protein and ATP plus L-malate. In principle, the only nucleotide formed should be AMP, which was expected to covalently bound to p45. Side reactions by impurities could form ADP, especially the kinases, shown to be active in the sample (see Figs. 39-40 & 42). The amount of AMP after hydrolytic cleavage of p45∼AMP (that was expected to be hydrolytically labile) should be small. Addition of L-malate had revealed in Fig. 32 to induce a turnover of ATP, that could

have resulted in AMP by forming dimers and oligomers of L-malate. Also, it was expected from published literature that p45∼AMP reacted with ATP to form AP4A (McLennan, 1992).

Thus, we expected a time dependent increase of AMP, ADP and AP4A. Time dependent reactions were monitored by TLC. The routine TLC system consisted of silica gel with a fluorescent indicator (Merck). The chromatography was developed by the method described by Guranowski et al., 2000.

Two experiments were carried out in parallel to determine the effect of incubation time on TLC resolution.

First experiment was conducted under the same condition as used for the formation of p45∼[³²P]AMP in the presence of L-malate, varying reaction times from 0 to 60 min at 25^oC.

The reaction mixture cocktail contained ligase buffer, 5 µCi of [α-³²P]ATP (3,000 Ci/mmol, Amersham Corp.), 2 mM ATP and 1 mM of L-malate in each of the eppendorfs. At 10 min intervals, 66 µg of the concentrated Toyopearl 650-M fraction was added to each eppendorf totaling a volume of 100 µl and incubated at 25^oC. At given times, 10 µl was removed quickly from each reaction mixture, stopped by adding 10 µl of 50 mM of ATP, and spotted onto a TLC plate (see chapter 2.2.14). After chromatography, the plate was then exposed to autoradiography, the results shown in Fig. 51. The experiment was also conducted with a single reaction mixture, and 10 µl samples were removed at ambient times and the reaction stopped by the addition of 10 µl of 50 mM of ATP. The TLC and autoradiograph were developed and the result was obtained in Fig. 52.

Incubation time (min) 0 10 20 30 40 50 60

← Front

← AMP

← AppppA

← ADP

← ATP

← Origin

Fig. 51: Effect of incubation time on TLC resolution. Lanes 1 to 7 refer to reactions incubated in parallel at 25^oC at different time periods ranging from 0 to 60 min. 1 µl samples corresponding to each incubation time were removed from the reaction mixtures at ambient times from different reaction mixtures, and spotted on the TLC-plate. The autoradiography was carried out for 4 days at -80^oC. The spots encircled in lane 8-11 indicate results obtained with non-labeled nucleotides (1 pmole of each nucleotides was spotted).

Incubation time (min) 0 10 20 30 40 50 60

← Front

← AMP

← AppppA

← ADP

← ATP

← Origin

Fig. 52: Effect of incubation time on TLC resolution. Lanes 1 to 7 refer to a single reaction incubated at 25^oC and samples were drawn at different times from 0 to 60 min. Conditions are otherwise the same as in Fig. 51.

Comparing Figs. 51 and 52, the autoradiographs show virtually the same results.

AMP, ADP, and AP4A were formed during the course of the reaction.

The results were as expected, confirming the formation of adenylate, the reaction of adenylate with L-malate to form AMP, and with ATP to form AP4A (Guranowski et al., 2000). The high rate of ADP formation was in agreement with the assumption of kinases.

The result on the formation of free AMP together with the kinetics for p45∼AMP observed in the presence of 10 mM L-malate (Fig. 32), suggested the formation of dimers or oligomers of L-malate. Attempts to resolve such products by TLC-system employed several methods to stain dimers or oligomers of L-malate. These systems involved vanillin (Adamson et al., 1999), iodine vapor (Zakrzewski and Ciesielski, 2002), or permanganate (Zakrzewski and Ciesielski, 2002). However, sucrose contained in the concentrated protein samples interfered with the detection.

3.11 Attempts to demonstrate the formation of dimers and oligomers of malic acid by employing L-[¹⁴C]malic acid and thin layer

chromatography

The kinetics shown in Fig. 32, depicting the transient formation of p45∼[³²P]AMP in the presence of 10 mM L-malate, suggested the formation of dimers and/or oligomers of malic acid. In the following experiment, it was attempted to identify such oligomers by employing

L-[¹⁴C]malic acid and TLC. Fig. 53 shows the results. Radioactivity runs close to the solvent front. Oligomers were expected to migrate less far. However, little or no evidence is seen in Fig. 53 of material that shows low mobility. As the concentration of L-[¹⁴C]malate was of the order of 18 µM (1 µCi reaction) and 90 µM (5 µCi reaction), very little formation of radioactively labeled oligomer expected in comparison with 10 mM L-malate in the kinetic study of Fig. 32. It was possible that the small amounts escaped the detection. Future experiments should apply high L-malate concentration. Since under such conditions TLC-plate detection was technically not possible, product analysis should be carried out by HPLC.

← Front

← Origin

0.2 µCi 1 µCi 1 µCi 5 µCi

Control Reaction

1 2 3 4

Fig. 53: Attempt to identify dimers and/or oligomers by employing L-[¹⁴C]malic acid and TLC. Lanes 1 and 2 refer to the control, which are 0.2 µCi and 1 µCi of L-[¹⁴C]malic acid, respectively, directly applied to TLC. Lanes 3 and 4 refer to the reaction mixtures which were carried out in the presence of 1 µCi and 5 µCi of L-[¹⁴C]malic acid, respectively. The autoradiography was carried out for 14 days at -80^oC.

3.12 Product analysis of the malate activation reaction by reversed phase