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Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

Bottom Line: Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex.We conclude that B plays a structural role in the plasma membrane.Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

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High-voltage paper electrophoresis of radiolabelled RG-II and the effect of boric acid, Pb2+ and glycosylinositol phosphorylceramides (GIPCs). (a) [3H]RG-II was loaded on to the paper after 9 h of pre-treatment of monomeric [3H]RG-II in 250 mm pyridine buffer, pH 4.7, with or without 1 mm H3BO3 or 1 mm H3BO3 plus 0.5 mm Pb(NO3)2. After electrophoresis, the paper was cut into strips, which were assayed for tritium by scintillation-counting. The positions of non-radioactive markers (glucose, galacturonic acid and Orange G) are also shown. (b) Samples were loaded on the paper after 4 h of pre-treatment of monomeric [3H]RG-II (RGIIm) in 50 mm ammonium acetate, pH 4.8, with or without various combinations of 1.2 mm boric acid (B), 0.5 mm Pb(NO3)2 (Pb2+), GIPCs (purified as described in Figure3), commercial phytoceramide (PC), and 0.1 m hydrochloric acid (HCl). After electrophoresis, papers were read on a LabLogic AR2000 radio-TLC Imaging Scanner. The distance migrated (d) is given relative to dRGIIm (the distance migrated by monomeric RG-II, that is to say approximately 6 cm). Grey bands indicate the positions of monomeric RG-II (right) and the GIPC–RG-II complex (left).
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fig04: High-voltage paper electrophoresis of radiolabelled RG-II and the effect of boric acid, Pb2+ and glycosylinositol phosphorylceramides (GIPCs). (a) [3H]RG-II was loaded on to the paper after 9 h of pre-treatment of monomeric [3H]RG-II in 250 mm pyridine buffer, pH 4.7, with or without 1 mm H3BO3 or 1 mm H3BO3 plus 0.5 mm Pb(NO3)2. After electrophoresis, the paper was cut into strips, which were assayed for tritium by scintillation-counting. The positions of non-radioactive markers (glucose, galacturonic acid and Orange G) are also shown. (b) Samples were loaded on the paper after 4 h of pre-treatment of monomeric [3H]RG-II (RGIIm) in 50 mm ammonium acetate, pH 4.8, with or without various combinations of 1.2 mm boric acid (B), 0.5 mm Pb(NO3)2 (Pb2+), GIPCs (purified as described in Figure3), commercial phytoceramide (PC), and 0.1 m hydrochloric acid (HCl). After electrophoresis, papers were read on a LabLogic AR2000 radio-TLC Imaging Scanner. The distance migrated (d) is given relative to dRGIIm (the distance migrated by monomeric RG-II, that is to say approximately 6 cm). Grey bands indicate the positions of monomeric RG-II (right) and the GIPC–RG-II complex (left).

Mentions: Using this extract, we investigated the ability of GIPC to bind RG-II by testing the effect of GIPC on the mobility of radiolabelled RG-II on paper electrophoresis. We incubated tracer quantities of [3H]RG-II with an excess of the purified GIPC under conditions suitable for RG-II dimerization (Chormova et al., 2013). On paper electrophoresis at pH 2.0, monomeric [3H]RG-II and the same preparation partially or fully dimerized by H3BO3, without or with 0.5 mm Pb2+ respectively, all migrated approximately 8 cm towards the anode (Figure4a). Thus dimeric RG-II had approximately 1.6× the charge of monomeric RG-II [estimated by application of Offord’s law, which states that mobility on paper electrophoresis is proportional to the Q:Mr2/3 ratio, where Q is the molecule’s net charge and where the molecular weight to the power of 2/3 is an indication of the molecule’s surface area (Offord, 1966; Fry, 2011)]. Co-migration of monomeric and dimeric RG-II is confirmed in Figure4b-ii,iii).


Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

High-voltage paper electrophoresis of radiolabelled RG-II and the effect of boric acid, Pb2+ and glycosylinositol phosphorylceramides (GIPCs). (a) [3H]RG-II was loaded on to the paper after 9 h of pre-treatment of monomeric [3H]RG-II in 250 mm pyridine buffer, pH 4.7, with or without 1 mm H3BO3 or 1 mm H3BO3 plus 0.5 mm Pb(NO3)2. After electrophoresis, the paper was cut into strips, which were assayed for tritium by scintillation-counting. The positions of non-radioactive markers (glucose, galacturonic acid and Orange G) are also shown. (b) Samples were loaded on the paper after 4 h of pre-treatment of monomeric [3H]RG-II (RGIIm) in 50 mm ammonium acetate, pH 4.8, with or without various combinations of 1.2 mm boric acid (B), 0.5 mm Pb(NO3)2 (Pb2+), GIPCs (purified as described in Figure3), commercial phytoceramide (PC), and 0.1 m hydrochloric acid (HCl). After electrophoresis, papers were read on a LabLogic AR2000 radio-TLC Imaging Scanner. The distance migrated (d) is given relative to dRGIIm (the distance migrated by monomeric RG-II, that is to say approximately 6 cm). Grey bands indicate the positions of monomeric RG-II (right) and the GIPC–RG-II complex (left).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: High-voltage paper electrophoresis of radiolabelled RG-II and the effect of boric acid, Pb2+ and glycosylinositol phosphorylceramides (GIPCs). (a) [3H]RG-II was loaded on to the paper after 9 h of pre-treatment of monomeric [3H]RG-II in 250 mm pyridine buffer, pH 4.7, with or without 1 mm H3BO3 or 1 mm H3BO3 plus 0.5 mm Pb(NO3)2. After electrophoresis, the paper was cut into strips, which were assayed for tritium by scintillation-counting. The positions of non-radioactive markers (glucose, galacturonic acid and Orange G) are also shown. (b) Samples were loaded on the paper after 4 h of pre-treatment of monomeric [3H]RG-II (RGIIm) in 50 mm ammonium acetate, pH 4.8, with or without various combinations of 1.2 mm boric acid (B), 0.5 mm Pb(NO3)2 (Pb2+), GIPCs (purified as described in Figure3), commercial phytoceramide (PC), and 0.1 m hydrochloric acid (HCl). After electrophoresis, papers were read on a LabLogic AR2000 radio-TLC Imaging Scanner. The distance migrated (d) is given relative to dRGIIm (the distance migrated by monomeric RG-II, that is to say approximately 6 cm). Grey bands indicate the positions of monomeric RG-II (right) and the GIPC–RG-II complex (left).
Mentions: Using this extract, we investigated the ability of GIPC to bind RG-II by testing the effect of GIPC on the mobility of radiolabelled RG-II on paper electrophoresis. We incubated tracer quantities of [3H]RG-II with an excess of the purified GIPC under conditions suitable for RG-II dimerization (Chormova et al., 2013). On paper electrophoresis at pH 2.0, monomeric [3H]RG-II and the same preparation partially or fully dimerized by H3BO3, without or with 0.5 mm Pb2+ respectively, all migrated approximately 8 cm towards the anode (Figure4a). Thus dimeric RG-II had approximately 1.6× the charge of monomeric RG-II [estimated by application of Offord’s law, which states that mobility on paper electrophoresis is proportional to the Q:Mr2/3 ratio, where Q is the molecule’s net charge and where the molecular weight to the power of 2/3 is an indication of the molecule’s surface area (Offord, 1966; Fry, 2011)]. Co-migration of monomeric and dimeric RG-II is confirmed in Figure4b-ii,iii).

Bottom Line: Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex.We conclude that B plays a structural role in the plasma membrane.Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

Show MeSH
Related in: MedlinePlus