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Abstract— The distribution of a series of enzymes in the post‐nuclear supernatant of rat brain homogenates was investigated following continuous density‐gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.

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Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. L-serine was also converted to pyruvate at about twice the racemization rate. L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site.