Partial Purification of Digitonin-Solubilized β-Glucan Synthase from Red Beet Root

Abstract

An enriched glucan synthase fraction was obtained from red beet root microsomes by sequential extraction with the detergents 3-1(3-cholamidopropyl)dimethylammoniol-l-propanesulfonate and digitonin. The digitonin suspension was centrifuged on a glycerol gradient, where a glucan synthase peak with a specific activity of 30-to 40-fold over microsomes was recovered. Most protein contaminants were found in the gradient pellet. The glucan synthase-containing fraction was largely free of plasma membrane and tonoplast-derived ATPase activity and was enriched with a protein subunit of 68 kilodaltons.Membrane-bound f,-glucan syntheses are a class of enzymes believed to be important in cell wall biosynthesis. Over the last several years, a great deal of effort has been directed towards understanding the properties and functions of these enzymes. Most work with glucan syntheses has been performed using isolated membrane fractions from various plant and microbial sources (5, 9,16,27,28, 30).To date, glucan syntheses have proven to be extremely difficult to purify. One problem has been stability in detergent (2,12,13,21,26 Membrane Isolation. Microsomes were isolated as previously described (8, 30).Glucan Synthase Assay. Glucan synthase assays were conducted in 100 ul assay mixtures containing 1 mm UDP-['4C] glucose (0.1 mCi per mmol), 5 mM MgCl2, 5 mm cellobiose, and 50 mM Tris-HCl (pH 7.0). The level of protein used varied between 2 and 50 tig, depending on the relative purity of the sample to be assayed. The mixtures were incubated at 30°C for 5 min and the reactions were then terminated by heating at 90 to 100°C for 10 min. Glucose incorporation was measured by the method of Smith and Stone (1975) as follows: Each reaction mixture was spotted on a Whatman GF/A filter disk (2.4 cm diameter), dried, and placed in a culture tube.The tubes were placed on ice and washed successively with 10 ml of 66% (w/v) ethanol containing 0.85 mm EDTA, 10 ml of 66% (w/v) ethanol, and 10 ml of 70% ethanol. Filters were rinsed with acetone, air dried, and counted. Quenching was corrected by counting known amounts of UDP-['4C]glucose that had been spotted on filters.Protein Determinations. Protein concentrations were measured by the Coomassie blue protein assay (7).Extraction of Microsomes with Digitonin. To solutions containing microsomes (0.85-2.0 ml) at protein concentrations of 2.4 to 5.3 mg per ml, an equal volume of a buffer containing 2% (w/v) digitonin, 5 mM MgCl2, and 50 mM Tris-HCl (pH 7.0) was added dropwise with stirring. The Glycerol Density Gradient Centrifugation. Linear gradients were made by combining 1.75 ml each of 12% (w/w) and 60% (w/w) glycerol in 5 mM MgCl2, 0.1 % digitonin, and 50 mM Tris-HCI (pH 7.0) on a gradient maker. In one experiment (Fig. 1), a www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-β-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment

“…This phenomenon is due to nonspecific precipitation of the enzyme as the combined result of a decrease in detergent levels and an increase in divalent cation concentration. It has already been shown that divalent cations inhibit CS solubilization (8,23) and that the enzyme solubilized with digitonin sediments faster in glycerol gradients containing cations than it does in the presence of chelators (12). Here we show that CS solubilized in CHAPS and enriched by product entrapment is also prone to cation-induced aggregation (Fig.…”

UDP-Glucose: (1,3)-β-Glucan Synthase from Daucus carota L.

“…At 4"C, the microsomal enzyme maintained full activity. Solubilized enzyme stored at -80'C began to show a decline in activity after 8 h and at 4"C after 4 h. Thus, solubilized carrot glucan synthase is considerably less stable than beet (11,12,28).…”

Cellulose and Callose Biosynthesis in Higher Plants (I. Solubilization and Separation of (1->3)- and (1->4)-β-Glucan Synthase Activities from Mung Bean)

“…It has been shown that glucan synthases are large protein complexes (>450 kD) and that several protein subunits ranging from 18 to 115 kD might be involved in glucan synthesis (Delmer, 1987;Eiberger and Wasserman, 1987; -uv Read and Delmer, 1987;Bulone et al, 1990;Lin et al, 1990). Efforts have been directed at defining the relationship between the individual components in various synthase preparations and, in particular, at identifying which polypeptides might participate in (1-»3)-and/or (1-»4)-/3-glucan synthesis.…”