Apamin is a neurotoxic polypeptide of known structure isolated from bee venom. Shuba and coworkers have recently shown that it abolishes the hyperpolarising action of externally-applied ATP on visceral smooth muscle (guinea pig stomach and taenia coli) as well as the hyperpolarisation (inhibitory junction potential) that follows stimulation of the non-adrenergic inhibitory nerve supply to these tissues. As it has been proposed that ATP is the neurotransmitter involved in the latter response, Vladimirova and Shuba tentatively concluded that apamin is a specific postsynaptic blocking agent of this non-adrenergic, possibly 'purinergic', inhibition. We have confirmed the important observation that nanomolar concentrations of apamin reduce inhibition by ATP and by non-adrenergic nerve stimulation, but further experiments suggest that, rather than acting as a specific blocker of ATP receptors, apamin inhibits the increase in potassium permeability caused by a number of agents, including ATP.

The free amino acid pool contents of Gram-negative bacteria (Aerobacter aerogenes, Erwinia carotovora, Pseudomonas fluorescens) were studied as functions of the growth environment and were compared with those from correspondingly grown cultures of Gram-positive bacteria (Bacillus subtilis var. niger, B. megaterium, B. polymyxa) and the yeast Saccharomyces cere visiae.Although the pools of the Gram-positive bacteria and the yeast contained five to 20 times the concentration of free amino acids present in the pools of Gram-negative bacteria, all pools were similar in containing only a limited range of detectable amino acids. Glutamate invariably predominated and generally accounted for over 50 % of the total amino acid content of the pool.The contents and composition of pools from micro-organisms maintained in steady states in chemostat cultures did not vary with time, but changed significantly with changes in either growth rate or the nature of the growth limitation. However, these pool variations were small compared with those resulting from addition of 2 % (w/v) NaCl to a culture of growing bacteria.With cultures of Gram-negative bacteria, sudden changes in medium salinity effected marked and rapid changes in free glutamate content; with Grampositive bacteria, similar changes occurred, but extremely slowly. Addition of 4 % (w/v) NaCl to growing yeast cultures brought about no observed changes in pool size or composition. These results are discussed with reference to the involvement of free amino acids in synthesis and functioning of microorganisms.

S U M M A R YThe enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.3. I . 2), glutamate synthase (Lglutamine : a-oxoglutarate amino transferase) and glutamate dehydrogenase (EC I . 4 . I .4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slowgrowing R. japonicum and R. lupini.Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M . sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system. I N T R O D U C T I O NThe assimilation of ammonia by bacteria proceeds via either glutamate dehydrogenase or the glutamine synthetase/glutamate synthase system, depending upon the organism and the ammonia concentration of the environment (Tempest, Meers & Brown, 1973; Brown, Macdonald-Brown & Meers, 1974). Dainty (1972) reported the presence of glutamate synthase in a strain of Clostridium pasteurianum which lacked glutamate dehydrogenase, while Nagatani, Shimizu & Valentine (1971) demonstrated the presence of glutamate synthase in a range of nitrogen-fixing bacteria and in bacteroids of Rhizobium japonicum prepared from Glycine max (soya bean) root nodules. Nagatani et al. (1971) claimed that the glutamine synthetase/glutamate synthase system was operative in ammonia assimilation

SUMMARYAmmonia-limited Aerobacter aerogenes, Erwinia carotovora, Pseudomonas jluorescens, Bacillus subtilis and B. megaterium synthesized glutamate from NH, and a-oxoglutarate by a process that involved first the synthesis of glutamine and then the reductive transfer of the glutamine amide-nitrogen to the a-position of a-oxoglutarate. The latter step required the recently reported enzyme ' glutamine(amide) : a-oxoglutarate amino-transferase oxido-reductase (NADP) ', some further properties of which are described here. This enzyme, from different organisms, always had a well-defined maximum activity at a pH value between 7.5 and 8.0; it had an apparent K, for 2-oxoglutarate between 0-1 and 2.0 mM and an apparent K, for glutamine between 0-2 and 1.8 mM. Glutamate (the metabolic end-product) and Mg2+ strongly inhibited the enzyme from Gram-negative bacteria but less so that from Grampositive species. Synthesis of glutamate by this enzyme required NADPH, and NADH was inactive ; pyruvate, oxaloacetate, a-oxobutyrate and 2-oxoisovalerate could not substitute for a-oxoglutarate, nor could the requirement for glutamine be met by asparagine, citrulline, arginine or urea. Although conditions that favoured the synthesis of this enzyme generally also favoured synthesis of glutamine synthetase and caused suppression of glutamate dehydrogenase formation, a close correlation between the bacterial contents of these different enzymes was not apparent.

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