1. 1. An isotopic, chromatographic assay for acetyl-CoA synthetase (acetate:CoA ligase (AMP), EC 188.8.131.52) has been developed which is both sensitive and rapid to perform. 2. 2. The rat brain synthetase activity behaves the same as the synthetase activity from other sources when exposed to increasing concentrations of Na+, K+, or NH4+. 3. 3. The subcellular location of the rat brain synthetase is primarily mitochondrial, but 13% of the activity was found in the cytoplasm. 4. 4. Kinetic studies on the rat brain synthetase preparation indicated higher substrate affinities than previously reported and marked substrate inhibition which occurred at moderate substrate concentrations. 5. 5. In view of the differences in substrate affinities and substrate inhibition of the rat brain acetyl-CoA synthetase compared to the synthetases from other sources, it is suggested that the results of comparative studies be interpreted cautiously.
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