No abstract

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.

The present account contains a brief summary of the concept of the glucose fatty acid cycle and of the experimental evidence on which it is based and of its possible importance in the interpretation of metabolic disturbances in obesity and maturity onset diabetes. Much of the experimental evidence on

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.