γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.

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y-Glutamylmethylamide (y-GMA) synthetase was detected in crude extracts of Methylophaga sp. AA-30, but neither methylamine dehydrogenase nor^V-methylglutamate dehydrogenase was observed. A large amount of y-GMA was accumulated in the cells when the growth on methanol-methylamine was inhibited with iodoacetate, but the accumulation was not observed in the cells grown on methanol-(NH4)2SO4. It is thought that y-GMAis a metabolic intermediate of the methylamine-dissimilating pathway in the bacterium. In addition, y-GMA-dissimilating enzymes were found in methylamine-grown cells. The enzymes, which consisted of H protein and L protein, required a-ketoglutaric acid, Mg2+ or Mn2+, and ammoniaas a co factor. Although the enzyme catalyzed the formation of glutamate from y-GMA,it did not catalyze the formation of /V-methylglutamate. Consequently, in this bacterium, methylamine seems to be metabolized through a different pathway from the TV-methylglutamate pathway.

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In the previous report, the authors clarified the distribution of nitrogen fixing bacteria in the sea water of coastal regions. The present paper deals with the occurrence of the bacteria in the water of open oceans, and in the bottom sediments of sea water environments .The standing crop of nitrogen fixing bacteria was quite small in the surface waters of the Mid-Pacific Ocean and the Japan Sea