PURIFICATION OF 1,3-BETA-D-GLUCAN SYNTHASE ACTIVITY FROM PEA TISSUE -2 POLYPEPTIDES OF 55 KDA AND 70 KDA COPURIFY WITH ENZYME-ACTIVITY

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.