Activity of glutamine synthetase (EC 6.3.1.2) in the soluble protein fraction of nodules from 21-day- old lupin plants was measured. On a fresh tissue weight basis, activity was approximately 500 times higher than in the soluble protein fraction from roots of inoculated plants and 300 times higher than in the bacteroid soluble protein fraction. The activity of glutamine synthetase in the bacteroids was approximately 10 times lower than in cultured rhizobia. Glutamine synthetase activity in the nodule cytoplasm was found to increase during nodule development over a time course which followed the induction of leghaemoglobin and nitrogenase. Glutaminase activity, which could be inhibited by L-5-diazo-4-oxo-norvaline, was detected in extracts of bacteroids and cultured rhizobia. The glutamine synthetases present in cultured rhizobia and in the nodule cytoplasm had different electrophoretic mobilities on polyacrylamide gels.

No abstract

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1–153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.