Increased selectivity in the detection of glycoproteins on nitrocellulose membranes by washing with sodium hydroxide solution

Kondo, Maki; Harada, Hiroshi; Sunada, Saburo; Yamaguchi, Takashi

doi:10.1002/elps.1150120918

Cited by 21 publications

(13 citation statements)

References 9 publications

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“…For sugar staining of protein bands, proteins in the SDS-polyacrylamide gel were transferred to an Immobilon-P membrane (Millipore). The glycoprotein blots were visualized by methods reported previously (21,22) using a kit (G. P. Sensor, Seikagaku Corp., Tokyo, Japan) following the manufacturer's recommendations.…”

Section: Protein Chemical Analysesmentioning

confidence: 99%

UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

Sawada

Suzuki

Ichimaida

et al. 2005

Journal of Biological Chemistry

107

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In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDPglucuronate to the 2؆-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6؆-O-malonylglucoside with a k cat value of 34 s ؊1 at pH 7.0 and 30°C. BpUGAT was highly specific for cyanidin 3-O-glucosides (e.g. K m for cyanidin 3-O-6؆-O-malonylglucoside, 19 M) and UDP-glucuronate (K m , 476 M). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28 -40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.

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Section: Protein Chemical Analysesmentioning

confidence: 99%

UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

Sawada

Suzuki

Ichimaida

et al. 2005

Journal of Biological Chemistry

107

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“…Proteins were stained for the carbohydrate content in situ according to the method of Kondo et al (1991). Briefly, SDS-PAGE and immunoblotting were performed as described above, and membranes were then washed in TTBS for 40 min at room temperature.…”

Section: Carbohydrate Stainingmentioning

confidence: 99%

Antigenically stable 35 kDa outer membrane protein ofSalmonella

Jaradat

Zawistowski

1998

Food and Agricultural Immunology

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Outer membrane protein (OMP) extracts from Salmonella species representing seven serogroups were examined by SDS-PAGE followed by immunoblotting. Two major proteins with apparent molecular weight of 24 and 35 kDa were identified. The latter protein was present only in Salmonella. Immunoblotting with a monoclonal antibody (MAb) 1D6 (IgA) demonstrated the 35 kDa OMP contains an antigen common for all tested Salmonella species with the exception of atypical species such as S. arizona. The type of growth media had no effect on the antigenicity of 35 kDa protein. However, the electrophoretic appearance of the 35 kDa protein was influenced by heat-treatment. Analysis of OMP extracts by SDS-PAGE and immunoblotting without heat-treatment revealed that MAb 1D6 bound several isoforms of this protein, a major form at 28 kDa and about eight minor forms in the range between 34 and 40 kDa. None of these forms contained carbohydrate moieties that may be responsible for the polymorphic appearance of the protein. These forms were converted to a single form by heat-treatment at 100°C indicating that the 35 kDa OMP is most likely a heat-modifiable protein. Furthermore, extended heattreatment (121°C, 15 min) did not affect the antigenicity of the 35 kDa OMP. Electron microscopy studies revealed that the 35 kDa OMP is exposed on a surface of the bacterial cells, although the frequency of epitopes recognized by the Mab 1D6 was low as can be inferred from the low number of gold particles attached to the cells.

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“…The polyadenylation signal (AATAAA) is present in nucleotides 1327-1332. There is a potential asparagine-linked glycosylation site conforming to the consensus sequence of Asn-X-Ser at amino acid positions 228 -230, although no glycosyl residues were detected in the purified GlcNAc 2-epimerase, when assayed by the method of Kondo et al (27). N-terminal amino acid was not detectable by Edman method (23).…”

Section: Resultsmentioning

confidence: 94%

Molecular Cloning and Identification of -Acyl-D-glucosamine 2-Epimerase from Porcine Kidney as a Renin-binding Protein

Maru¹,

Ohta²,

Murata

et al. 1996

Journal of Biological Chemistry

119

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Increased selectivity in the detection of glycoproteins on nitrocellulose membranes by washing with sodium hydroxide solution

Cited by 21 publications

References 9 publications

UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

Antigenically stable 35 kDa outer membrane protein ofSalmonella

Molecular Cloning and Identification of -Acyl-D-glucosamine 2-Epimerase from Porcine Kidney as a Renin-binding Protein

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