Enzymology and molecular biology of cell wall biosynthesis. Progress report Page: 4 of 11
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of several to many other polypeptides (which most authors have interpreted as multiple
suhunits of the GS-II system) made it difficult to assign activity unambiguously to this
polypeptide. Because of this, plus concern that more than one polypeptide might occur in
the 55 kDa band which could lead, via an irrelevant amino acid sequence, to cloning an
erroneous gene, we set out to iry to purify GS-II as completely as possible.
The purification attempts required mass scale preparation of plasma membranes
from pea stems. We optimized the aqueous polymer two-phase partition system for
isolating plant plasma membranes such that we can prepare 0.12 g of highly purified
plasma membranes from 600 g of 8-da old pea stems in one day. Two to three such
preparations are then used for digitonin solubilization of GS-II, followed by the product
entrapment procedure. This single step gives a --200 fold purification of GS-II. Further
fractionation by preparative isoelectric focusing and by glycerol gradient centrifugation
increases the purification to -350 fold over the specific activity of GS-II in the plasma
membrane. In the highly purified preparations only two polypeptides, of 55 and 70 kDa,
occur. Immunoblotting shows that the 55 is the same polypeptide that we previously
reported, based on partial purification and immunological evidence, is involved in GS-II
activity. This is the first time that GS-II has been purified to this extent. The key to this
purification was finding that after isolation by product entrapment, GS-II activity can be
stabilized by substantially lowering the buffer and detergent concentrations in the media.
The detailed data on purification are presented in a manuscript (Dhugga & Ray, 1993), in
preparation for submission to I. Biol. Chem., the current draft of which is appended
hereto.
Our attempts at directly sequencing the 55 and 70 kDa polypeptides failed,
apparently because these polypeptides are N-terminally blocked. We subsequently
separated tryptic peptides derived from the 55 and 70 kDa polypeptides on a C-4 HPLC
column (equipment that we do not possess, but have kindly been allowed the use of by the
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Ray, P. M. Enzymology and molecular biology of cell wall biosynthesis. Progress report, report, March 20, 1993; California. (https://digital.library.unt.edu/ark:/67531/metadc1310443/m1/4/: accessed June 6, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.