Glycoprotein Biosynthesis in Cotyledons of <i>Pisum sativum</i> L

Beevers, Leonard; Mense, Rose M.

doi:10.1104/pp.60.5.703

Cited by 44 publications

(33 citation statements)

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“…11 and 21). Such compounds have been identified in developing pea (2) and french bean (9,12,13) cotyledon tissue. Dolichyl phosphate has been purified from soybean and shown to act as an effective acceptor for glycosyl transfer from sugar nucleotides (5,22).…”

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Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Bailey

Deluca²,

Dürr³

et al. 1980

Plant Physiol.

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Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-E-16_3Hlglucosamine and guanosine-5'-diphospho-n-IU-14Clmannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations. (2) and french bean (9, 12, 13) cotyledon tissue. Dolichyl phosphate has been purified from soybean and shown to act as an effective acceptor for glycosyl transfer from sugar nucleotides (5,22).Evidence that lipid-linked oligosaccharides can be assembled in a sequential manner from UDP-GlcNAc and GDP-mannose by membrane preparations from developing soybean cotyledons and that some of these, as in mammalian systems, may be involved in the synthesis of endogenous storage glycoproteins is presented here. Earlier studies in these laboratories have been concerned mainly with the assembly of glycolipids in growing tissue (pea stem) where glycoproteins do not accumulate (1, 10), and it was of particular interest to employ similar techniques with a tissue where a major part of metabolism is geared to the synthesis and deposition of glycoprotein. MATERIALS AND METHODSPlant Material. Soybean (Glycine max. var. Prize) was grown in Vermiculite with a 12-h photoperiod at 28 C (daytime) and 22 C (night). Plants were inoculated with Rhizobium japonicum and watered with N-free medium as described previously (27). Cotyledons were harvested when their fresh weight was between 100 and 250 mg.Membrane Isolation and Incubation. A membrane preparation was obtained from developing soybean cotyledons by modification of the method previously used in studies with pea stem tissue (1). The cotyledons were homogenized with a mortar and pestle at 4 C in 0.1 M Tris-HCl (pH 7.4) containing 5 mm dithioerythritol, 10 mM MgCl2 and 0.4 M sucrose, and the brei was filtered through Miracloth (Calbiochem). The filtrate was centrifuged at low speed (lOOOg for 10 min) and the pellet was dispersed for 20 s using a tissue homogenizer (Polytron, Brinkmann Instruments, at halfmaximum force). The homogenate was recentrifuged (lOOOg for 10 min) and the supernatant was combined with that from the previous centrifugation. A total membrane preparation was obtained from this supernatant by centrifugation at high speed (48,000g for 60 min).

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confidence: 99%

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Bailey

Deluca²,

Dürr³

et al. 1980

Plant Physiol.

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“…Particulate fractions isolated from developing pea cotyledons can transfer labeled sugar from nucleoside diphosphate sugars to lipid components and to components which remain in the residue following lipid extraction (2,17). Earlier repots have indicated a preferential stimulatory effect by Mg24 and Mn2" on the transfer of radioactivity from GDP-[14CJMan2 to lipid intermediates and glycoproteins (4,12,13,15,18,21).…”

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“…Incubation was at 37 C for 60 min with the reaction terminated by the addition of 0.7 ml of H20 and 2.5 ml of chloroform-methanol (2:1, v/v). Further lipid extraction and analysis of the lipid-free residue were described previously (2).…”

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“…Furthermore, a UDP-GlcNAc transferase was localized in RER membranes isolated from pea cotyledons (17 vested and the testa and embryos were removed. The crude particulate fraction was prepared as described by Beevers and Mense (2). Optimal Mg24 and Mn2" requirements for transferase activity in this fraction were determined by incubating 150 ,ul of the pellet suspension with 100 ,Il of 50 mm Tris-HCl (pH 7.0) containing 10 mM KCI, 5 mm 2-mercaptoethanol, 2 ,ul of '4C-labeled nucleoside diphosphate sugar, and 48 1A of H20 or 48 ,ul of a MgCl2 or MnCl2 solution.…”

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“…The crude particulate fraction was prepared as described by Beevers and Mense (2). Optimal Mg24 and Mn2" requirements for transferase activity in this fraction were determined by incubating 150 ,ul of the pellet suspension with 100 ,Il of 50 mm Tris-HCl (pH 7.0) containing 10 mM KCI, 5 mm 2-mercaptoethanol, 2 ,ul of '4C-labeled nucleoside diphosphate sugar, and 48 1A of H20 or 48 ,ul of a MgCl2 or MnCl2 solution. The specific activities of the labeled nucleoside diphosphate sugars are given below.…”

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Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum

Nagahashi¹,

Mense²,

Beevers³

1978

Plant Physiol.

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In crude particulate fractions isolated from pea (Pisum sativum) cotyledons, the transfer of radioactivity from GDP-I14Cimannose to glycolipid appears to be preferentially stimulated by Mn2' while the transfer to lipidfree residue is enhanced by Mg2'. In contrast, the transfer of radioactivity from UDP-N-acetyl-I14Clglucosamine to glycolipid shows preferential stimulation by Mg2" while the transfer to lipid-free residue prefers Mn2". These results are accounted for by the differential stimulation by Mg24 and Mn24 of glycosyl transferases associated with subcellular membranes which were separated by isopycnic sucrose density centrifugation.Particulate fractions isolated from developing pea cotyledons can transfer labeled sugar from nucleoside diphosphate sugars to lipid components and to components which remain in the residue following lipid extraction (2, 17). Earlier repots have indicated a preferential stimulatory effect by Mg24 and Mn2" on the transfer of radioactivity from GDP-[14CJMan2 to lipid intermediates and glycoproteins (4,12,13,15,18,21 nucleoside diphosphate sugars are given below. Incubation was at 37 C for 60 min with the reaction terminated by the addition of 0.7 ml of H20 and 2.5 ml of chloroform-methanol (2:1, v/v). Further lipid extraction and analysis of the lipid-free residue were described previously (2).Sucrose Gradients. Membrane fractions used in linear sucrose density gradients were prepared by sedimenting particulate material from 250 to 13,000g and 13,000 to 40,000g (17). The crude pellets were either washed in GM (0.5 M sucrose + 5 mM 2-mercaptoethanol + 30 mm Tris-MES [pH 7.5]) or GM + 5 mM EDTA and pelleted at the initial force (17). Crude pellets were overlaid on identical gradients (15-60%/o sucrose, w/w) and centrifuged to equilibrium at 81,000g for 15 hr. Preparation of sucrose gradients and fractionation were described elsewhere (17).Enzyme Assays for Isolated Membrane Fractions. Inosine diphosphatase (IDPase) activity of pH 7.5 was determined as described by Leonard and VanDerWoude (14). NADH-Cyt c reductase activity was assayed spectrophotometrically by following the reduction of Cyt c at 550 nm (10).Glycosyl transferase activities were determined either by measuring incorporation of label from nucleoside diphosphate sugar into trichloroacetic acid-insoluble material or by assaying incorporation into lipid intermediates and lipid-free residues using an extraction procedure described earlier (2). Although we use the singular form of GDP-Man transferase and UDP-GlcNAc transferase when we speak of trichloroacetic acid-precipitable material, it should be kept in mind that these precipitates represent an aggregate of glycosyl transferase activities. UDP-N -acetyl-[U-14C]glucosamine (300 mCi/mmol, 10 ,uCi/4001,) and GDP-[U-'4C]mannose (179 mCi/mmol, 50 ,Ci/2 ml) were used as substrates to measure activities of UDP-GlcNAc and GDP-Man transferases. Membrane fractions (0.3 ml) were incubated with 0

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The assembly of lipid‐linked oligosaccharides in plant and animal membranes

et al. 1979

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Membrane preparations from growing regions of pea stems and actively-dividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono- and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9--12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(14C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl-(14C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(14C)mannose alone. Incubation of the membranes with GDP-(14C)mannose in the presence of Mn2+, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.

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Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Cited by 44 publications

References 31 publications

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum

The assembly of lipid‐linked oligosaccharides in plant and animal membranes

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