Glycerol kinase and dihydroxyacetone kinase in rat brain.

The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to ,sn-glycerol-3phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat hrain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined, Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45nmol of glycerol was phosphorylated/min per rng of protein. The K , for glycerol was 7 0 ~ ~ at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K, for dihydroxyacetone was 0.6 mM. Glycerol kinase activity was also present in the cytoplasm of brain. However, the spccific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 100). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2-3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K , for dihydroxyacetone was 6 0 ~ ~ . This cytosol fraction was also found to phosphorylate u-glyccraldehyde and L-glyceraldehyde at a rate of 3&400/, to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed. GLYCEROL kinase (ATP: glycerol-3-phosphotransferase EC 2.7.1.30) catalyzes the phosphorylation of glycerol by AI'P to sn-glycerol-3-phosphate (G-3-P). This enzyme was discovered by KALCKAR (1939) in kidney. The properties of the enzyme were first described in detail by BUBLITZ & KENNEDY (1954); WIELAND & SUYTER (1957). Using an enzymatic assay, WIELAND & SUYTER (1957) concluded that this kinase activity is present only in liver and kidney but not in other mammalian organs. However, later workers using more sensitive assays showed the presence of this enzyme in a wide variety of tissues (for a review see THORNER & PAULUS, 1973). To datc glycerol kinase has not been described in the brain, although there is indirect evidence to suggest its presence in that organ. A number of workers showed that when radioactive glycerol was injected intracranially into rats it was incorporated into the brain glycerolipids (HOKIN & HOKIN, 1958; LAPETINA ef aL, 1969; BENJAMINS & MCKHANN, 1973; OBRIEN & GEISON. 1974). This suggests that glycerol is phosphorylated in brain to G-3-P and subsequently acylated and converted to different lipids (KENNEDY, 1962).