In the preceding paper (Schumacher, Smith & Williams, 1965) it was shown that thalidomide was converted in the body into a number of compounds, several of which are derivatives of the amino acids, glutamic acid and glutamine. Since thalidomide is racemic, some of its breakdown products will, therefore, contain amino acid residues of the unnatural D-series. Any of the biological effects (embryotoxic, neurotoxic and sedative) of thalidomide could be due to the drug or its metabolites interfering with the normal metabolism of L-glutamine and L-glutamic acid. These amino acids are prominently involved in the metabolic activity of the central and peripheral nervous system (Tower, 1960), although their role in foetal development is not clear. There are indications, however, that they may be involved in morphogenesis (Deuchar, 1962).In this paper the effects of thalidomide and its hydrolysis products on three enzymes concerned with glutamate metabolism (namely glutamine synthetase, glutamate decarboxylase and glutamate dehydrogenase), on the embryos of pregnant rabbits and on the hexobarbitone-induced sleeping time in rats, will be described.
METHODSGlutamine synthetase. An acetone-dried powder of rat brain was prepared. Brains from freshly killed adult female rats were rapidly removed, cooled on ice, weighed and homogenized in ten volumes of ice-cold acetone. The resulting suspension was centrifuged at 4,000 revs/min for 10 min and the acetone was poured off. The solid residue was resuspended in a further ten volumes of ice-cold acetone and centrifuged as before. The acetone layer was again decanted and the solid residue was dried at 40 C in vacuo over silica gel and paraffin wax. To prepare an extract containing glutamine synthetase activity, the acetone-dried powder (0.5 g) was homogenized for 2 min in 10 ml. 0.1 M-tris-potassium chloride buffer (pH 7.3) at 00 C using a Waring blender. The mixture was centrifuged at 4,000 revs/min for 5 min and the supernatant fluid was used as a source of glutamine synthetase activity, which was estimated by the method of Rudnick, Mele & Waelsch (1955). A typical incubation mixture contained the following: sodium L-glutamate (100 pmoles), adenosine triphosphate (10 jmoles), magnesium chloride (40 pmoles), 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride (tris) (100 jmoles), potassium cyanide (80 pmoles), hydroxylamine (40 Mmoles) and the enzyme solution (0.3 ml.) in a final volume of 2 ml. Substances to be tested for inhibitory activity were dissolved in the tris buffer. The low solubility of thalidomide precluded it from being tested at the same concentration as those used for other compounds. Hydroxylamine was freshly prepared before use by neutralizing hydroxylamine hydrochloride with 2 N-sodium hydroxide. The preparations were incubated at 370 C for 20 min and then the reaction was stopped by adding 1.5 ml. of a solution containing equal volumes of 5% (w/v) ferric chloride (in 0.1 N-hydrochloric acid), 2.5 N-hydrochloric acid and 15% (w/v) trichloracetic acid. The react...