A method for the solubilization and reconstitution of red beet (1,3)-t-D-glucan synthase with the detergent 3-(3-cholamidopropyl) dimethylammoniol-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations nhibitory. A preextraction of membranes with 03% CHAPS and 5 millimar Mg2e prior to the soubilization step was found to remove protein con ats and inrease the specific activity of the solubilized enzyme. Conditios for recovering activity from Sepharose 4B gel filtration cohmns were defined. Additio of phospholipids and low levels of CHAPS in column elution buffers resulted in complete fimctional reconstitution with 100% recovery of added activity. Specific activities were increased 20-to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.tempts to further purify the enzyme in digitonin were unsuccessful. This may be due to the complex micellar nature ofdigitonin, which has a critical micelle concentration of0.02% and a micellar mol wt of 70,000 (15). It is probable that the difficulty in purifying glucan synthase arises from the fact that each proteindetergent mixed micelle contains a heterogeneous population of solubilized proteins. Therefore, we began to investigate the ability of other detergents to solubilize glucan synthase such as Triton X-100, which removed phospholipid but did not solubilize the enzyme (26). Morrow and Lucas (19) described the solubilization of sugar beet petiole glucan synthase with zwittergent 3-14; however, specific activities were low relative to microsomes suggesting stability problems. This paper describes the solubilization of red beet root glucan synthase with CHAPS,4 a zwitterionic derivative of cholic acid (14). The hydrophobic cholic acid portion of CHAPS bears structural similarity to the digitogenin portion ofdigitonin. The major advantages ofthis detergent over digitonin include a relatively high CMC (0.6% versus 0.02%), dialyzability, and a much lower micellar mol wt (6,500 versus 70,000). In addition, we demonstrate the reconstitution of CHAPS-solubilized glucan synthase into phospholipid vesicles by gel filtration chromatography and define conditions for maximizing activity recovery.Understanding the biosynthesis of cell wall macromolecular components has attracted considerable interest (7,8,25 Glucan synthase from red beet storage tissue has proven to be a good candidate for purification. Microsomal and plasma membrane preparations contain high levels of activity (27) and the enzyme is relatively stable (9). Like other -(41,3)-E-glucan synthases it is readily solubilized by digitonin (9, 10). The digitonin solubilized enzyme was purified up to 40-fold from microsomal membranes by glycerol density gradient centrifugation (10). At-'

No abstract

UDP-glucose:(1,3)-j%-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A2. Enzyme activity could not be restored to the phospholipasetreated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.

The membrane-bound UDP-glucose-fl-(1, branes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 40C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.3Biosynthesis of plant cell wall polysaccharides is generally believed to be mediated by glycosyl transferases localized at '

16,19,[24][25][26][27]Isotope analysis of plant matter provides a powerful method of studying photosynthetic modes. In addition to the well known separation of C3 plants from C4 and CAM plants based on stable carbon isotope ratios (17), oxygen and hydrogen isotope ratios ofcellulose and cellulose nitrate, respectively, also are influenced by photosynthetic mode (20,(22)(23)(24)(25). Analysis of C3, C4, and CAM plants growing in the vicinity of each other show that cellulose nitrate from CAM plants is enriched in deuterium relative to C3 and C4 plants (20, 22, 25). Stemnberg et al. (20, 22-25) concluded that the difference in 6D values between CAM plants and C3 and C4 plants are due to fractionations occurring during biochemical reactions particular to CAM plants. C4 plants also tend to have higher abundances of deuterium and '80 than C3 plants (20,22,27). Stemnberg et al. (22)