Role of lipid in the cellulose synthetase enzyme system from oat seedlings

Pinsky, A.; Ordin, Lawrence

doi:10.1093/oxfordjournals.pcp.a074463

Cited by 19 publications

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“…DISCUSSION This study demonstrates that glucan synthase activity can be restored to Triton X-100-treated membrane fractions by the addition of phospholipids. These results support previous assumptions (17,22) and provide additional evidence that glucan synthase activity is strongly influenced by its membrane environment.…”

Section: Resultssupporting

confidence: 91%

“…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.…”

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confidence: 89%

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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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Section: Resultssupporting

confidence: 91%

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confidence: 89%

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

Wasserman¹,

McCarthy²

1986

Plant Physiol.

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“…The gradients were centrifuged at 100,000g for 90 min, and the fractions were prepared for electron microscopy and assayed as for procedure I. Glucan Synthetase Assay. Glucan synthetase activity was determined by the procedures of Ordin and Hall (31) (33).…”

Section: Methodsmentioning

confidence: 99%

β-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem

et al. 1974

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Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. l8-1,3-and p-1,4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 uM) UDP-glucose concentrations synthesis of alkaliinsoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mM) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of g-1,4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, ,-1,4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of 6-1 ,3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations.In vivo studies of cell wall biogenesis indicate that cellulose synthesis occurs at the cell surface, possibly at the surface of the plasma membrane (28,34,39,44,52), while pectic and hemicellulosic polysaccharides are synthesized by the Golgi apparatus (1,5,10,18,27,40). In contrast, Ray et al. (36) found UDP-glucose: /3-1,4-glucan glucosyltransferase (3-1,4-glucan synthetase) activity to be localized in a membrane frac-

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“…This is probably among the reasons why activation is not obtained in vitro by simply adding IAA or other auxins to isolated particles, nor in vivo by treatment with IAA in the cold (37 (4,14,24,58) in which this effect is due to formation of deoxyhexose nucleotides (13,17,27,58 In view of the effect of sugar supply on glucan synthetase activity, I considered whether the activating effect of IAA could be due simply to the fact that IAA promotes uptake of sugars by pea stem segments (1). This relatively trivial explanation is ruled out by the experiment of Table IV (3,32,41,50,53), and change in permeability of the membrane particles to substrate (35).…”

Section: Data Inmentioning

confidence: 99%

Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

Ray

1973

Plant Physiol.

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The 2-to 4-fold rise in particle-bound 3-glucan synthetase (uridine diphosphate-glucose:,6-1 ,4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca'+ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by a, a'-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.Under conditions previously defined (37), treatment of pea stem tissue with IAA or other auxins causes a rapid, temperature-dependent rise in the level of particle-bound /8-glucan synthetase activity (UDP-glucose:/8-1 ,4-glucan glucosyltransferase) obtainable from the tissue. This report considers how this hormonal effect depends upon cellular metabolism. A somewhat similar investigation of the effect of IAA, applied in lanolin paste, on GDP-glucose-dependent glucan synthetase activity in stem tissue of whole decapitated pea seedlings was published recently (48). METHODSPlant material and glucan synthetase preparation and assay were as described previously (37). Briefly, pea stem segments 8 mm long cut from the region of maximal elongation rate were kept in a moist atmosphere at 35 C for 2.5 to 3 hr to reduce glucan synthetase activity substantially below the initial level; then a medium containing usually 30 mm sucrose was added and incubation at 35 C was continued for 1 hr. Then the tissue was treated with plus-or minus-IAA media at a specified temperature. In the case of inhibitor experiments the media also contained 16 mm K phosphate buffer, pH 6.1, and 'Supported by a grant from the National Science Foundation. when necessary to ensure establishment of the appropriate inhibition by the time of addition of IAA the sucrose pretreatment media also contained the inhibitor, as specified for the experiments reported. At the end of the treatment period the tissue was chilled on ice, washed with ice water, and used for preparation and assay of glucan synthetase (37).In Vivo Isotope Incorporation Experiments. Samples of 50 segments were incubated in 3.0 ml of labeled substrate in 16 mM K phosphate buffer, pH 6.1, on a reciprocating shaker (120 oscillations/min) at 25 C. At the conclusion of incubation the segments were washed thoroughly with ice water. For assay of incorporation from sugars into particles and cell wall the segments were ground in the same manner (37) as for glucan synthetase assay; the cell wall residue (1,OOOg) and particle pe...

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Role of lipid in the cellulose synthetase enzyme system from oat seedlings

Cited by 19 publications

References 0 publications

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

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

β-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem

Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

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