Research Report Dr

Research Report Dr. W. Michalke

Universitt Freiburg
Institut fr Biologie III
Schnzlestrasse 1
D-79104 Freiburg i. Br.
Germany

e-mail: wolfgang.michalke@biologie.uni-freiburg.de

MABs against plant PM-H⁺-ATPase

Monoclonal antibodies directed to plasma membrane H⁺-ATPase have been generated, using purified protein from Zea mays coleoptiles as antigen (Ltzelschwab, Ph.D. thesis, Freiburg 1990). These antibodies preferentially recognize cells in the vascular bundles and stomatal guard cells of Zea mays shoots and Cucurbita pepo hypocotyls. The antibodies also preferentially recognize one of the two cell layers in leaves of Elodea canadensis (Ltzelschwab, Ph.D. thesis, Freiburg 1990, Baur et al., 1996, Bot Acta 109, 382-387).
Almost no immunofluorescence could be detected in Elodea stems, whereas the leaves were obviously labelled by H⁺ATPase antibodies, markedly more immunofluorescence was seen in the layer of smaller cells on the lower side of the leaves (Bauer et al. 1996, Bot Acta 109, 382-387). In parallel experiments using microsomes isolated separately from elodea leaves and stems, only the leaf microsomes contained ATPase activity with properties of the plasma membrane H⁺-ATPase, while microsomes derived from Elodea stems hardly any activity of this type was detected. In western blots of leaf microsome preparations a 100 kDa band was detected by anti-ATPase antibody, whereas only a very faint band was detected in microsome preparations from Elodea stems. Top

2. Polypeptides detected by anti-plasma membrane H⁺-ATPase antibodies in lower plants

Monoclonal antibodies directed to maize plasma membrane H⁺-ATPase and antiserum directed to oat plasma membrane H⁺-ATPase were used to detect antigenic molecules in several species of algae. Mikrosomes prepared from algae were screened for the presence of H⁺-ATPase antigen using SDS-PAGE and western blotting. In none of the microsomal preparations from the algae (20-40 g protein per lane) was a signal of comparable strength encountered, as in Cucurbita hypocotyl membrane vesicles (4 g per lane) used as positive control. Polypeptides with a molecular weight around 100 kDa were detected by anti-ATPase antiserum and monoclonal anti-ATPase antibodies in microsomes from the green algae Spirogyra, Mougeoutia, and Zygnema.
Polypeptides with a molecular weight at 66 kDa were labelled in microsomes from Chara with monoclonal anti-ATPase antibodies as well as with anti-ATPase antiserum. This polypeptide was however of extreme abundance (strong Coomassie stain!) in the Chara microsomes, so the labelling by anti-ATPase antibodies was not of very high specificity. Even carefully monitored inhibition of proteases in Chara microsomes did not yield a labelled polypetide in the range of 100 kDa. Polypeptides in microsome preparations from other green as well as brown and red algae species did show no, or very weak and variable labelling with anti-ATPase antibodies. Microsomes from Lunularia (a liverworth) did show clearly a 100 kDa polypeptide labelled, but again distinctly weaker as in the Cucurbita control. Top

Acknowledgement

The experimental work in this report has been financially supported by the Land Baden-Wrttemberg.

Publications 2001-2005