PREPARATION AND CHARACTERIZATION OF SEALED INSIDE-OUT PERIBACTEROID MEMBRANE-VESICLES FROM PISUM-SATIVUM L AND GLYCINE-MAX L ROOT-NODULES BY AQUEOUS POLYMER 2-PHASE PARTITIONING
Jh. Christiansen et al., PREPARATION AND CHARACTERIZATION OF SEALED INSIDE-OUT PERIBACTEROID MEMBRANE-VESICLES FROM PISUM-SATIVUM L AND GLYCINE-MAX L ROOT-NODULES BY AQUEOUS POLYMER 2-PHASE PARTITIONING, Journal of plant physiology, 147(2), 1995, pp. 175-181
A method is described for the preparation of sealed inside-out peribac
teroid membrane (PBM) vesicles from pea (Pisum sativum L.) and soybean
(Glycine max L.) root nodules by aqueous polymer two-phase partitioni
ng. At optimal conditions about one-fourth of the total membrane prote
in 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 meas
uring the activities of marker enzymes for the cytoplasmic surface (AT
Pase (EC 3.6.1.3) and 1,3-beta-glucan synthase (GS II; EC 2.4.1.34)) w
ith 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 nod
ule PBM is compared with that of the plasma membrane from uninfected p
ea roots. The GS II activity of PBM constitutes 10% of that in pea roo
t plasma membrane whereas ATPase activity of PBM amounts to 70% of tha
t in pea root plasma membrane. The pea PBM ATPase resembles the plasma
membrane-type ATPase on the basis of insensitivity to nitrate, stimul
ation by NH4+ and sensitivity to vanadate. The results reveal that hig
h purity preparations of inside-out sealed PBM vesicles are obtained w
ithout contamination from bacteroid or plasma membrane by the present
procedure. Thus, preparations suitable for studies on transport and si
gnal transduction mechanisms across the PBM from the bacteroid side ar
e now available.