Solubilization of a digitonin-stable glucan synthase from red beet root

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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

Section: Resultsmentioning

confidence: 70%

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

Eiberger

Wasserman²

1987

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“…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).…”

Section: Stability Of the Solubilized Enzymementioning

confidence: 94%

“…Red beet storage tissue has been an attractive source of this enzyme since large quantities of membranes are readily prepared and the enzyme possesses good stability (12). Although significant progress has been made toward its purification, its subunit composition has not yet been confirmed.…”

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confidence: 99%

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

Lawson¹,

Mason²,

Sabin³

et al. 1989

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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 '

“…At levels of 0.5% or above, those typically used for glucan synthase solubilization (1, 8-10, 13, 14, 21), only 30% of the original activity remained. Since digitonin has no adverse effects on red beet root glucan synthase in untreated membranes (8), these results suggest that Triton X-100 is removing some component, possibly boundary phospholipid, that enhances the enzyme's stability in digitonin.…”

Section: Resultsmentioning

confidence: 77%

“…Glucan synthase from red beet root was shown to be stable for a week in digitonin (8) and more recently a fraction enriched 30-to 40-fold in specific activity over microsomes was isolated by glycerol gradient centrifugation of a digitonin extract (LL Eiberger et al, submitted for publication). Since digitonin has an extremely low critical micelle concentration (15), it was of interest to look at the behavior of glucan synthase in other detergents.…”

mentioning

confidence: 99%

Regulation of Plasma Membrane β-Glucan Synthase from Red Beet Root by Phospholipids

Wasserman¹,

McCarthy²

1986

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Extraction of red beet root plasma membranes with the detergent Triton X-100 at a level of 2.0% (weight/volume) resulted in the depletion of over 90% of total membrane phospholipid and the reduction of glucan synthase activity by 80 to 90%. Reconstitution of the delipidated Triton X-100, 100,OOOg fraction in the presence of phospholipids restored glucan synthase activity. The most effective phospholipid was phosphatidylethanolamine, which restored 110 to 144% of the original activity at 0.5% (weight/volume). Glucan synthase in the phospholipid-reactivated Triton X-100-treated fraction was enriched 9-fold in specific activity relative to microsomal membranes but was unstable in digitonin. These results support the hypothesis that glucan synthase activity is regulated by its phospholipid environment.Glucan synthases are a group of extensively studied higher plant membrane enzymes thought to be important in the biosynthesis of cell wall polysaccharides. Yet, due to the fact that no glucan synthase has ever been purified to homogeneity, our understanding of the regulation of cell wall polysaccharide biosynthesis has remained severely limited. Since #-glucan synthase is believed to be an integral membrane protein, it has been proposed that phospholipids might modulate its activity. Evidence to support this possibility has been presented by Pinsky and Ordin (22) and more recently by Kauss and Jeblick (17), who demonstrated that glucan synthase from suspension cultured soybean is inhibited by a range of amphipathic molecules in the presence of digitonin.The nonionic detergent digitonin has been widely utilized to solubilize glucan synthases (1, 8-10, 13, 14, 21 examined. It was previously reported that Triton X-100 treatment of glucan synthase-containing membranes results in significant activity losses (9,13,14). It is demonstrated here that these activity losses may have been a result of the removal of phospholipid from the complex and that red beet root glucan synthase requires a phospholipid environment for activity.MATERIALS AND METHODS Materials. Red beets were obtained from local markets. The tops were removed and roots were stored in moist vermiculite for up to 3 weeks at 4°C until use. UDP-('4C]glucose (220 mCi/ mmol) was purchased from ICN Radiochemicals, Irvine, CA. Unlabeled UDPG,2 digitonin, and phospholipids were products of Sigma Chemical Co. L-a-Lecithin from soybean was obtained from Calbiochem. Triton X-100 was obtained from Rohm and Haas. Phospholipid dispersions were prepared by evaporation of chloroform, followed by resuspension in a 70 ,L of a solution containing 0.5 ,umol cellobiose, 0.5 IUmol MgCl2, and 5 imol Tris-HCI (pH 7.0). Tubes were then subjected to repeated sonication until uniform suspensions were obtained. Color reagents for protein assays were purchased from Bio-Rad. Digitonin solutions were prepared by dissolving digitonin in 5 mM MgC92, 50 mM Tris-HCl (pH 7.0), and stirring under heat until turbidity had disappeared. The solutions were then cooled on ice until use.Membrane I...