Measurement of binding affinity of various metal elements ... - CiteSeerX

RIKEN Review No. 35 (May, 2001): Focused on New Trends in Bio-Trace Elements Research

Measurement of binding affinity of various metal elements to plant apoplast protein in root tips Jun Furukawa,∗ Rieko Hirunuma, Shuichi Enomoto, and Tomoko M. Nakanishi∗ Radioisotope Technology Division, Cyclotron Center, RIKEN

We measured the affinity of 13 metal elements to a plant apoplast protein. The apoplast protein was incubated with a multitracer in the presence or absence of Al and then ultrafiltrated through a molecular membrane (10,000 M.W.). Based on the gamma-ray spectra of the membranes, the binding affinity of elements was analyzed. At pH 7.0, seven elements (Be, Fe, Zn, Sr, Y, Zr, and Ru), whereas at pH 6.0, five elements (Be, Zn, Sr, Y, and Zr), showed a relatively high affinity to the protein. Beryllium and Zr exhibited similar binding characteristics to the membrane and the protein at both pH 6.0 and pH 7.0. When Al was added, the binding affinity of Be and Zr to the protein decreased, suggesting that there was competition between Al and either Be or Zr for binding to the apoplast protein.

Introduction The acid-soil Al ion has been reported as one of the main factors that inhibits plant growth, therefore limiting crop production. 1) Aluminum toxicity was found to inhibit root growth rapidly. Aluminum causes general toxic symptoms that are similar to nutrient deficiencies,2,3) but these symptoms appear to be the consequences of root growth inhibition caused by the selective action of Al in root tips.4) Many research studies targeting various micro organs (chromatin, Golgi body, plasma membrane, vacuole, and cell wall) of the cell have been reported over a long time, 5–9) but the most critical sites of Al toxicity are not yet determined. Horst 10) suggested the sensitive binding sites of Al in apoplast and that competition for these binding sites might be responsible for root growth inhibition during short-term Al treatment. However, the knowledge on Al toxicity to the plasma membrane or cell wall is very limited. Moreover, in the case of the other component of apoplast, there is very little information on the Al effect. In this study, we focused on the binding affinity of various metal elements to an apoplast protein in the presence of Al by means of the multitracer technique.

Experimental A multitracer was prepared from silver foil irradiated with a N beam of 135 MeV/nucleon at the RIKEN ring cyclotron. The group separation of typical elements and REEs was performed following the method reported by Liu et al .11)

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Protein extraction from apoplast in root tips Two-day-old seedlings [Glycine max. (L.) Merr. cv. Tsurunoko] were washed well with distilled water. After washing, root tips were excised between 2 and 5 mm from the root apex, corresponding to the elongation zone, i.e., without root caps. Excised root tips collected from about 200 seedlings were washed well with distilled water and then sam∗

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Graduate School of Agricultural and Life Science, University of Tokyo

ples were put in an ultrafree microtube with a filter, the pore size of which is 0.22 µm (Millipore Co., USA). Approximately 400 µl of the extraction buffer containing 50 mM MgCl2 was added into the microtubes for infiltrated extraction. Then, the packed tissues were placed under vacuum for 5 min and the microtubes were centrifuged for 5 min at 2000 g. Vacuum infiltration and centrifugation were performed by the same method reported by Kataoka et al . 12) The collected infiltrate was immediately mixed with 50 µl of the protein stabilization solution, 0.5 M MES buffer (pH 6.0) containing 10 mM PMSF and 0.5 M DTT, or 0.5 M MOPS buffer (pH 7.0) containing the same concentrations of PMSF and DTT, and then ultrafiltrated through a molecular membrane (Millipore Co., USA, 10000 M.W.). Measurement of the binding affinity of multitracer to apoplast protein A portion of the infiltrate was used for protein determination using the Bio-Rad Protein Assay kit (Richard, CA, USA). Each aliquot (0.2 ml) was mixed with 0.2 ml of 0.05 M MES (pH 6.0) or MOPS (pH 7.0) buffer solution containing the multitracer. The measurement of the binding affinity of the multitracer to the apoplast protein was carried out following the method reported by Sotogaku et al .13) Samples were incubated for 30 min at 4◦ C and then ultrafiltrated through a molecular membrane (10000 M.W.). The gamma-ray spectra of the membranes and filtrate were measured using a pure Ge detector, and were compared with those of the standard multitracer solution. To determine the Al effect on the metal binding affinity to the apoplast protein, buffer solutions (pH 6.0 and 7.0) containing the multitracer and AlCl3 (1 µM) were prepared.

Results and discussion From the gamma-ray spectroscopy analysis, 13 elements (Be, Na, Sc, Mn, Fe, Co, Zn, Se, Sr, Rb, Y, Zr, and Ru) were simultaneously determined. The results are given as percentages of the standard multitracer solution. Binding to the membranes at ultrafiltration Table 1 shows the radioactivity of each element remaining

Table 1. Radioactivity of elements that remained on a membrane (as a percentage, compared with that of the prepared multitracer solution). The abbreviation N.D. indicates that the peak was not detected by γ ray measurement. The Blank column indicates that there was only the multitracer in the buffer solution. The Al column indicates when 1 µM Al was added to the buffer solution. The Protein column and Al&Protein column indicate the cases with the buffer solution containing 1 µg protein and 1µg protein plus 1 µM Al, respectively. pH 6.0 Be Na Sc Mn Fe Co Zn Se Sr Rb Y Zr Ru

Blank 48.9 N.D. 81.0 7.3 73.8 N.D. N.D. 38.7 1.1 0.8 49.4 63.3 51.2

Al 77.2 N.D. 77.0 7.5 74.3 1.7 1.7 40.1 1.5 1.1 60.9 71.5 54.8

Protein 76.7 N.D. 77.7 6.5 71.6 1.7 2.7 39.8 2.3 1.5 79.8 71.1 56.5

Al&Protein 64.2 N.D. 76.7 7.3 73.7 1.6 3.0 38.3 2.3 2.0 75.2 63.7 54.9

on the membrane. As is shown in the column of the Blank, more than half of the Sc, Fe, Zr, and Ru amounts were found to bind to the membrane. The aim of this research is to determine the binding affinity of various metal elements to the apoplast protein in the presence of Al. Therefore, before applying the apoplast protein to the buffer solution, only Al was added to clarify the difference in the binding affinity of the multitracer to the membrane. When 1 µM Al was applied to the buffer solution, Be, Sr, Y, and Zr amounts in the membrane were increased at pH 6.0, whereas at pH 7.0, only the Be amount was found to increase significantly. Metal binding to protein When 1 µg of protein was applied to the buffer solution, the radioactivity of Zn, Sr, and Y on the membrane was increased to more than 30 percent at pH 6.0, whereas at pH 7.0, only that of Zn and Y as found to increase compared to those when only Al was added. The radioactivity values of Be and Zr showed that these two elements had similar characteristics in terms of binding to the membrane and the protein at both pH 6.0 and pH 7.0. In the presence of Al alone in the buffer solution, the amounts of Be and Zr that remained on the membrane were increased but the amounts were the same as those when the protein alone was added. However, in the presence of Al and the protein, amounts of both these elements on the membrane decreased, indicating that the binding affinity of these elements to the protein was reduced in the presence of Al. The decrease in the binding affinity suggested that there was a competition between Al and either Be or Zr for binding to the apoplast

pH 7.0 Be Na Sc Mn Fe Co Zn Se Sr Rb Y Zr Ru

Blank 60.1 N.D. 60.0 6.8 49.0 N.D. 11.6 29.6 1.3 1.0 61.0 42.6 49.5

Al 82.7 N.D. 54.5 7.7 54.3 N.D. 10.4 28.9 N.D. 0.9 57.2 48.2 44.2

Protein 82.4 N.D. 63.0 7.5 58.9 2.9 19.9 30.8 2.4 1.6 64.6 49.5 56.9

Al&Protein 84.1 N.D. 62.6 8.0 59.9 3.5 19.3 31.5 3.1 2.2 65.2 50.3 57.0

protein. At pH 7.0, similar effects due to the presence of both Al and the protein were not observed in any of the other tracers. In the case of Zn, there was no effect of Al on the amount of binding to the protein at either pH 6.0 or pH 7.0. Since this was the first trial, we would like to perform the same experiment again to determine the reproducibility of our results.

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