The Journal of Cell Biology, Vol 108, 2193-2200, Copyright © 1989 by The Rockefeller University Press

ARTICLES

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer

JP Gorvel, Z Mishal, F Liegey, A Rigal and S Maroux
Centre de Biochimie et de Biologie Moleculaire, Centre National de la Recherche Scientifique, Marseille, France.

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

• Genestier, L., Prigent, A.-F., Paillot, R., Quemeneur, L., Durand, I., Banchereau, J., Revillard, J. P., Bonnefoy-Berard, N. (1998). Caspase-dependent Ceramide Production in Fas- and HLA Class I-mediated Peripheral T Cell Apoptosis. J. Biol. Chem. 273: 5060-5066 [Abstract] [Full Text]  
• Massey-Harroche, D, Mayran, N, Maroux, S (1998). Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues. J. Cell Sci. 111: 3007-3015 [Abstract]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents