IMPROVED RECOVERY OF (1-]3),(1-]4)-BETA-D-GLUCAN SYNTHASE ACTIVITY FROM GOLGI-APPARATUS OF ZEA-MAYS (L) USING DIFFERENTIAL FLOTATION CENTRIFUGATION

The synthesis of (1-->3),(1-->4)-beta-D-glucan (MG) is associated with the Golgi apparatus of maize. Identification of in vitro reaction pro ducts by enzymic hydrolysis and separation of diagnostic oligosacchari des by HPLC was used as a specific assay for MG synthase activity. Lar ge quantities of highly enriched membrane are: needed to study the enz yme components of MG synthesis. We directly obtained highly enriched G olgi apparatus in a single flotation centrifugation, without the neces sity of an initial downward centrifugation. This new procedure has imp roved the yield of Golgi apparatus, and has improved recovery of MG sy nthase activity. The substrate in glucan synthase reactions is UDP-Glc , but UDP-Glc is also a substrate in many other reactions, including t he production of simple glucosides. In addition, much of the labeled G lc from UDP-Glc is broken down to Glc-I-P and Glc under MG synthase re action conditions. We have explored some inhibitors of phosphatase, ph osphorylase, phosphodiesterase, and glucosidase activities in order to minimize these competing reactions and increase the activity of MG sy nthase.