Ks. Dhugga et Pm. Ray, PURIFICATION OF 1,3-BETA-D-GLUCAN SYNTHASE ACTIVITY FROM PEA TISSUE -2 POLYPEPTIDES OF 55 KDA AND 70 KDA COPURIFY WITH ENZYME-ACTIVITY, European journal of biochemistry, 220(3), 1994, pp. 943-953
From pea plasma membranes isolated by aqueous polymer two-phase partit
ioning we have purified 1,3-beta-D-glucan synthase [glucan synthase-II
(GS-II) or callose synthase], an enzyme that several reports have sug
gested consists of between six and nine different subunits. The proced
ure involves (a) preliminary removal of peripheral proteins by 0.1% di
gitonin; (b) solubilization of GS-II with 0.5% digitonin; (c) precipit
ation of activity-irrelevant proteins from the digitonin extract by Ca
2+, spermine and cellobiose, which are GS-II effecters needed in step
(d); (d) product entrapment by formation of 1,3-beta-D-glucan from UDP
-Glc by GS-II in the presence of the mentioned effecters, followed by
centrifugal sedimentation of product micelles and elution of proteins
therefrom with buffer; (e) preparative isoelectric focusing (IEF) of p
roduct-entrapped proteins; and (f) glycerol gradient centrifugation of
the fractions of peak GS-II activity from IEF. The procedure yields 3
00-fold enrichment of GS-II specific activity over that in isolated pl
asma membranes, and 5500-fold over that in the original homogenate. Ou
t of approximately six principal polypeptides that occur after the pro
duct entrapment step, the glycerol gradient GS-II activity peak contai
ns only two major polypeptides, one of 55 kDa and another of 70 kDa, p
lus minor amounts of one or two others whose distribution and occurren
ce indicate are not responsible for GS-II activity. Antisera against e
ither the 55-kDa or the 70-kDa polypeptide adsorb more than 60% of the
GS-II activity from a product-entrapped preparation. After native gel
electrophoresis, GS-II activity is associated with a single protein b
and of very large molecular mass, whose principal components are the 5
5-kDa and 70-kDa polypeptides, accompanied by minor amounts of a few o
ther polypeptides most of which do not occur in enzyme prepartions pur
ified by the previously described procedure. The 55-kDa but not the 70
-kDa component can be labeled by ultraviolet irradiation of the plasma
membranes in the presence of [alpha-P-32]UDP-Glc under GS-II assay co
nditions. It seems likely, therefore, that the 55-kDa and 70-kDa polyp
eptides form a large catalytic complex of which the 55-kDa component i
s the UDP-Glc-binding subunit.