Glucan biosynthesis in red beet root microsomes and tissue slices

Wasserman, Bruce P.; Ventola, Carol L.; Eiberger, Laura L.

doi:10.1021/jf00061a040

Cited by 9 publications

(4 citation statements)

References 19 publications

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“…2, middle). These results are consistent with previous work, in which PM vesicles partitioned at 38% to 40% Suc, whereas tonoplast vesicles are found at 25% to 28% Suc (Poole et al, 1984;Wasserman et al, 1985).…”

Section: Membrane Distribution Of Pmip31 and Pmip27supporting

confidence: 93%

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Barone

Shih

et al. 1998

Plant Physiology

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Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfidelinked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.The PM of higher plants has been the subject of extensive research focusing on the areas of recognition, water and ion transport, signal transduction, and cell wall polymer bio-

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Section: Membrane Distribution Of Pmip31 and Pmip27supporting

confidence: 93%

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Barone

Shih

et al. 1998

Plant Physiology

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“…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).…”

mentioning

confidence: 99%

CHAPS Solubilization and Functional Reconstitution of β-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

Sloan¹,

Rodis²,

Wasserman³

1987

Plant Physiol.

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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-'

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“…It is currently not known whether the enzyme activity we are measuring is the one found in terminal complexes. In a previous study, it was shown that the reaction product of the microsomal system was mixed ,3-1,3 and ,8-1,4 glucan and not true cellulose (25). The question of whether glucan synthase activity measured in vitro is catalyzed by the same complex that synthesizes cellulose in vivo has been the subject of much speculation.…”

Section: Resultsmentioning

confidence: 99%

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

Wasserman¹,

McCarthy²

1986

Plant Physiol.

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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...

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Glucan biosynthesis in red beet root microsomes and tissue slices

Cited by 9 publications

References 19 publications

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

CHAPS Solubilization and Functional Reconstitution of β-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

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

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