Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays |
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Authors: | Martin Rössle Jürgen Kreusch Karl Decker |
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Institution: | (1) Biochemisches Institut der Albert-Ludwigs-Universität, Hermann-Herder-Str. 7, D-7800 Freiburg i. Br., Federal Republic of Germany;(2) Present address: Medizinische Universitätsklinik, Hugstetter Str. 55, D-7800 Freiburg i. Br., Federal Republic of Germany |
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Abstract: | CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as 1-14C]citrate formed in the presence of 4-14C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with 2,3-14C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE
dithioerythritol
- NEM
N-ethylmaleimide
- NES
N-ethylsuccinimide
Enzymes (EC 2.7.2.1)
Acetate kinase, ATP: acetate phosphotransferase
- (EC 3.1.3.1)
Alkaline phosphatase, orthophosphoric monoester phosphohydrolase
- (GOT)
Aspartate aminotransferase
- (EC 2.6.1.1)
L-aspartate:2-oxoglutarate aminotransferase
- (CS)
Citrate synthase
- (EC 4.1.3.7)
citrate oxaloacetate-lyase (pro 3S-CH2COOacetyl-CoA)
- (EC 2.8.3.8)
CoA-transferase, acyl-CoA:acetate CoA-transferase
- (EC 1.1.1.37)
Malate dehydrogenase, L-malate:NAD+ oxidoreductase
- (EC 1.18.1.3)
NADH:ferredoxin reductase, ferredoxin:NAD+ oxidoreductase
- (EC 3.1.4.1)
Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase
- (EC 2.3.1.8)
Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase
- (EC 2.3.1.9)
Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase
A preliminary account of this work has been given (Decker et al. 1976) |
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Keywords: | Clostridium kluyveri Acetyl-CoA content of C kluyveri CoASH content of C kluyveri Regulatory function of the acetyl-CoA/CoASH ratio Isotope assay of acetyl-CoA Quantitative determination of CoASH with 14C-N-ethyl-maleimide |
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