SOLUBLE AUXIN BINDING PROTEIN IN PEA?

PNL Volume 17 1985

RESEARCH REPORTS

Hajek, K. Institute of Genetics

University of Bonn, Federal Republic of Germany

As Dollstadt (1) already proved eight years ago, there are mem-

brane bound auxin binding proteins in pea. In 1981 Jacobsen (2)

showed the existence of auxin binding proteins in the cytoplasm of pea

epicotyls. The binding assay he used was the ammonium sulphate (AS)

precipitation. To confirm and to compare this method, some other

methods had been used and optimized. The soluble auxin binding pro-

teins (sABP) in the crude cytosol could be proved by three methods.

The PEI (polyethyleneimine)-treated filter test, used first by Bruns,

et al. (3) for estradiol receptors; a method using NC (Nitrocellu-

lose)-filters, which bind specifically proteins; and the AS-precipita-

tion method, all showed the existence of the sABP.

All calculations were carried out according to Scatchard (4).

To get the sABP more purified, chromatofocusing (CF) was used as

a separation method for proteins (5). The crude cytosol was intro-

duced into a column filled with a CG-gel and the different proteins

were eluted according to their pI, and characterized by their respec-

tive elution pH. The elution pattern which was obtained is shown in

Fig. 1. The pattern was reproducible. This fact and the composition

of the eluted protein peaks was checked by SDS-electrophoresis.

Most of the binding appeared in peak IV, which is a double peak

with a pH range betwen 5.4 and 6.1. In this protein fraction sABP

could be proved by the AS-precipitation method, the PEI-treated filter

test, and NC-filter test, and also with the equilibrium dialysis.

One complete run required up to four days, so it was obvious that

more satisfactory results would be obtained if first the duration of a

run could be shortened and second the resolution improved. This was

achieved using an HPLC-pump and a pressure resistant column. The

duration of one run could be shortened to one day and the double peak

IV was separated in two close but distinct peaks: peak IVa with pH 6.1

and peak IVb with pH 5.4 (Fig. 2).

The protein of the other peaks did not show any binding, but sABP

could be detected in the protein elution of peak IVa and peak IVb.

This could be shown by the AS-precipitation method, the PEI-treated

filter test, and the NC-filter test.

So there are at least two sorts of sABP in pea, which seem to be

time-dependent (6).

We intend to purify these most sensitive sABP in order to show

the influence on the gene activity in the cells of epicotyls from pea.

1. Dollstadt, R., K. Hirschberg, E. Winkler, and G. Hubnei. 1976.

Planta 130:105-111.

2. Jacobsen, H.-J. 1981. Cell Biol. Intern. Rep. 5:768.

3. Bruns, R., K. Lawson-Wendling, and T. Pugsley. 1983. Anal.

Biochem. 132:74-81.

4. Scatchard, G. 1949. Ann. N.Y. Acad. Sci. 51:660-672.

5. Sluyterman, L. A. 1.982. TIBS, May 1982

6. Jacobsen, H.-J. 1984. Plant and Cell Physiol. 25:867-873.



	PNL Volume 17 1985	RESEARCH REPORTS 23



	Fig. 1. Elution pattern from a separation of the crude cytosol by the CF-column. The pH was measured continuously. Proteins of peak V strongly bound to the matrix and have been eluted by 1M NaCl in elution buffer.



	Fig. 2. Elution pattern from a separation of the crude cytosol by the CF-column. The HPLC-pump and a pressure resistant column had been used. The pH was measured continously.



22	PNL Volume 17 1985	RESEARCH REPORTS

SOLUBLE AUXIN BINDING PROTEIN IN PEA? Hajek, K. Institute of Genetics University of Bonn, Federal Republic of Germany As Dollstadt (1) already proved eight years ago, there are mem- brane bound auxin binding proteins in pea. In 1981 Jacobsen (2) showed the existence of auxin binding proteins in the cytoplasm of pea epicotyls. The binding assay he used was the ammonium sulphate (AS) precipitation. To confirm and to compare this method, some other methods had been used and optimized. The soluble auxin binding pro- teins (sABP) in the crude cytosol could be proved by three methods. The PEI (polyethyleneimine)-treated filter test, used first by Bruns, et al. (3) for estradiol receptors; a method using NC (Nitrocellu- lose)-filters, which bind specifically proteins; and the AS-precipita- tion method, all showed the existence of the sABP. All calculations were carried out according to Scatchard (4). To get the sABP more purified, chromatofocusing (CF) was used as a separation method for proteins (5). The crude cytosol was intro- duced into a column filled with a CG-gel and the different proteins were eluted according to their pI, and characterized by their respec- tive elution pH. The elution pattern which was obtained is shown in Fig. 1. The pattern was reproducible. This fact and the composition of the eluted protein peaks was checked by SDS-electrophoresis. Most of the binding appeared in peak IV, which is a double peak with a pH range betwen 5.4 and 6.1. In this protein fraction sABP could be proved by the AS-precipitation method, the PEI-treated filter test, and NC-filter test, and also with the equilibrium dialysis. One complete run required up to four days, so it was obvious that more satisfactory results would be obtained if first the duration of a run could be shortened and second the resolution improved. This was achieved using an HPLC-pump and a pressure resistant column. The duration of one run could be shortened to one day and the double peak IV was separated in two close but distinct peaks: peak IVa with pH 6.1 and peak IVb with pH 5.4 (Fig. 2). The protein of the other peaks did not show any binding, but sABP could be detected in the protein elution of peak IVa and peak IVb. This could be shown by the AS-precipitation method, the PEI-treated filter test, and the NC-filter test. So there are at least two sorts of sABP in pea, which seem to be time-dependent (6). We intend to purify these most sensitive sABP in order to show the influence on the gene activity in the cells of epicotyls from pea. 1. Dollstadt, R., K. Hirschberg, E. Winkler, and G. Hubnei. 1976. Planta 130:105-111. 2. Jacobsen, H.-J. 1981. Cell Biol. Intern. Rep. 5:768. 3. Bruns, R., K. Lawson-Wendling, and T. Pugsley. 1983. Anal. Biochem. 132:74-81. 4. Scatchard, G. 1949. Ann. N.Y. Acad. Sci. 51:660-672. 5. Sluyterman, L. A. 1.982. TIBS, May 1982 6. Jacobsen, H.-J. 1984. Plant and Cell Physiol. 25:867-873.