Preparation and characterization of sealed inside-out peribacteroid membrane vesicles from Pisum sativum L and Glycine max L root nodules by aqueous polymer two-phase partitioning

A method is described for the preparation of sealed inside-out peribacteroid membrane (PBM) vesicles from pea (Pisum sativum L.) and soybean (Glycine max L.) root nodules by aqueous polymer two-phase partitioning. At optimal conditions about one-fourth of the total membrane protein from osmotically shocked symbiosomes is recovered as inside-out PBM vesicles. This corresponds to a yield of 25 to 40 mu g of PBM protein . g(-1) nodule. The sidedness of tight vesicles is determined by measuring the activities of marker enzymes for the cytoplasmic surface (ATPase (EC 3.6.1.3) and 1,3-beta-glucan synthase (GS II; EC 2.4.1.34)) with and without detergent. The difference between enzyme activity with and without detergent designates latency, and based on these results it is estimated that the preparations contain 70 to 80% sealed inside-out PBM vesicles. The specific activity of GS II and ATPase in pea nodule PBM is compared with that of the plasma membrane from uninfected pea roots. The GS II activity of PBM constitutes 10% of that in pea root plasma membrane whereas ATPase activity of PBM amounts to 70% of that in pea root plasma membrane. The pea PBM ATPase resembles the plasma membrane-type ATPase on the basis of insensitivity to nitrate, stimulation by NH4+ and sensitivity to vanadate. The results reveal that high purity preparations of inside-out sealed PBM vesicles are obtained without contamination from bacteroid or plasma membrane by the present procedure. Thus, preparations suitable for studies on transport and signal transduction mechanisms across the PBM from the bacteroid side are now available.