Affiliations: In Vitro Végétale, 59 Chemin des Mésanges,
CH 1225 Chêne-Bourg, Geneva, Switzerland | Department of Medicinal Chemistry and Molecular
Pharmacology, HANS Life Sciences Research Building, Purdue University, 201
South University Street, West Lafayette, IN 47907-2064, USA
Note: [] Address for correspondence: D.J. Morré, Department of
Medicinal Chemistry and Molecular Pharmacology, Purdue University, 201 S.
University Street, West Lafayette, IN 47907-2064, USA. Fax: +1 765 494 4007;
E-mail: morre@pharmacy.purdue.edu
Abstract: Growth hormone-responsive and nucleoside triphosphate-dependent
enlargement of inside-out vesicles of plasma membranes from soybeans prepared
by aqueous two-phase partition and everted by freezing and thawing has been
achieved in a cell-free system. In the presence of 100 μM
ATP in 40 mM HEPES buffer, pH 7, enlargement of isolated plasma membrane
vesicles was accelerated by the synthetic plant growth factor,
2,4-dichlorophenoxyacetic acid (2,4-D), compared to ATP alone, 2,4-D alone or
no additions. After 20 min with 1 μM 2,4-D, vesicles
increased in diameter, 20% on average. Although vesicle diameters in the
presence or absence of 2,4-D overlapped, the means were clearly separated. The
20% increases in diameter corresponded to a doubling of vesicle volume. Both
100 μM ATP and 1 μM 2,4-D were
necessary to stimulate the cell-free vesicle enlargement. In the presence of
1 μM 2,4-D, enlargement observed with 100 μM ATP was greater than with either 10 μM ATP or
500 μM ATP alone. In the presence of 100 μM ATP, vesicle enlargement was proportional to the logarithm of 2,4-D
concentration. With the growth-inactive 2,4-D analog, 2,3-D, no vesicle
enlargement was observed either alone or in the presence of 100 μM ATP. Right side-out vesicles did not enlarge in response to either
ATP, 2,4-D or the two in combination suggesting that the responsible ATP site
was on the inside of the cell.
