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.
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Abbreviations
- PAG:
-
protein A gold complex
- GARG:
-
antibodygold complex
- PBS:
-
phosphate buffered saline (50 mM sodium phosphate, 0.9% NaCl, pH 6.9)
References
Barker HA (1981) Amino acid degradation by anaerobic bacteria. In: Snell EE (ed) Ann Rev Biochem, vol 50. Palo Alto, pp 23–40
Buckel W (1980) The reversible dehydration of (R)-2-hydroglutarate to (E)-glutaconate. Eur J Biochem 106:439–447
Buckel W, Liedtke H (1986) The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols. Eur J Biochem 256:251–257
Buckel W, Semmler R (1983) Purification, characterization and reconstitution of glutaconyl-CoA decarboxylase, a biotin dependent sodium pump from anaerobic bacteria. Eur J Biochem 136:427–434
Buckel W, Dorn U, Semmler R (1981) Glutaconate CoA-transferase from Acidaminococcus fermentans. Eur J Biochem 118:315–321
Dimroth P (1987) Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria. Microbiol Rev 51:320–340
Mey J de, Moeremans M, Gemeus G, Moydens R, de Brabander M (1981) High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol Int Reports 5:889–899
Rohde M, Mayer F, Meyer O (1984) Immunocytochemical localization of carbon monoxide oxidase in Pseudomonas carboxydovorans. The enzyme is attached to the inner aspect of the cytoplasmic membrane. J Biol Chem 259:14788–14792
Rohde M, Johannssen W, Mayer F (1986) Immunocytochemical localization of the soluble NAD-dependent hydrogenase in cells of Alcaligenes eutrophus. FEMS Microbiol Lett 36:83–86
Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use protein A-gold complex. J Histochem Cytochem 26:1074–1081
Roth J, Bendayan M, Carlemalm E, Villinger M, Garavito M (1981) Enhancement of structrual preservation and immunocytochemical staining in low temperature embedded pancreatic tissuc. J Histochem Cytochem 29:663–669
Schweiger G, Buckel W (1984) Studies on the dehydration of (R)-2-hydroxyglutarate in Acidaminococcus fermentans. A radical mechanism? Arch Microbiol 137:302–307
Schweiger G, Dutscho R, Buckel W (1987) Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein. Eur J Biochem 169:441–448
Slot JW, Geuze HJ (1981) Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Ultrastruct Res 26:31–43
Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci 76:4350–4354
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Rohde, M., Mayer, F., Dutscho, R. et al. Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans . Arch. Microbiol. 150, 504–508 (1988). https://doi.org/10.1007/BF00422295
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DOI: https://doi.org/10.1007/BF00422295