Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans |
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Authors: | Manfred Rohde Frank Mayer Roland Dutscho Gert Wohlfarth Wolfgang Buckel |
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Institution: | (1) Institut für Mikrobiologie der Georg-August-Universität Göttingen, Grisebachstrasse 8, D-3400 Göttingen, Federal Republic of Germany;(2) Mikrobiologie, Fachbereich Biologie der Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-3550 Marburg, Federal Republic of Germany |
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Abstract: | We have investigated the in situ location of glutaconyl-CoA decarboxylase and 2-htdroxyglutaryl-CoA dehydratase in Acidaminococcus fermentans using the antibody-gold and protein A-gold techniques carried out as a post-embedding immunoelectron microscopic procedure. Polyclonal antisera were raised in rabbits against homogeneous fractions of the enzymes. Anaerobically grown cells of A. fermentans of the late exponential growth phase were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde (final concentrations) in the growth medium. Dehydration of the cells was achieved with methanol. The cells were embedded in the low temperature embedding resin Lowicryl K4M. The markers indicative for antigenic sites of the two enzymes unequivocally demonstrate that the sodium pump glutaconyl-CoA decarboxylase is located at the cell periphery being a membrane-bound enzyme as expected whereas 2-hydroxyglutaryl-CoA dehydratase is a soluble cytoplasmic enzyme.Abbreviations PAG
protein A gold complex
- GARG
antibodygold complex
- PBS
phosphate buffered saline (50 mM sodium phosphate, 0.9% NaCl, pH 6.9) |
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Keywords: | Acidaminococcus fermentans Glutamate fermentation Electron microscopy Immunocytochemistry Post-embedding labelling Antibody-gold complexes Protein A-gold complexes |
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