The proteins of rapeseed (<i>Brassica napus</i> L.) soluble in salt solutions

Bhatty, R. S.; McKenzie, S.; Finlayson, A. J.

doi:10.1139/o68-178

Cited by 87 publications

(50 citation statements)

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“…SDS-polyacrylamide gel electrophoresis, and amino acid composition studies confirm previous observations on rapeseed and other related species (3,5,11,12,14,15,19,21,26,27). However, the use of two-dimensional gel electrophoresis systems provides new information.…”

Section: Discussionsupporting

confidence: 83%

“…

(3,11,14,15,19,21In this paper, we characterize radish 12 and 1.7 S proteins and compare them to their rapeseed equivalents. SDS-polyacrylamide gel patterns and amino acid compositions are reported.

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confidence: 99%

“…Curiously, even in closely related, economically important species, such as Brassica and Sinapis, storage proteins have been very poorly characterized, so that little information is available about their complexity and structure when compared to the extensively studied legume or cereal storage proteins (4,28). Brassica, Sinapis, and radish storage proteins also accumulate in protein bodies (12,17 (3,11,14,15,19,21 Neuf) were ground in liquid N2 and extracted as described previously (5). The extract was fractionated into aliquots which were stored frozen at -80°C until use.…”

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confidence: 99%

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Characterization of Radish (Raphanus sativus) Storage Proteins

Laroche

Aspart²,

Delseny³

et al. 1984

Plant Physiol.

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(3,11,14,15,19,21In this paper, we characterize radish 12 and 1.7 S proteins and compare them to their rapeseed equivalents. SDS-polyacrylamide gel patterns and amino acid compositions are reported. In addition we have analyzed each family using two different two-dimensional gel electrophoresis systems. Combining isoelectrofocusing in the first dimension and SDS in the second, we demonstrated that the 12 S family is much more complex than previously assumed. Finally, using a second system in which the first dimension is run in nonreducing conditions whereas the second is in the presence of a reducing agent, we obtained information concerning the associations between the different polypeptides. MATERIALS AND METHODS Extraction and Purification of Storage Proteins. Radish seeds (cv Rond rose a bout blanc, Vilmorin) and rapeseeds (cv JetNeuf) were ground in liquid N2 and extracted as described previously (5). The extract was fractionated into aliquots which were stored frozen at -80°C until use. The 12 and 1.7 S storage protein particles were purified by gel filtration through a Sepharose 6B column (90 x 1 cm) equilibrated in 10 mm sodium phosphate, 0.5 M NaCl, pH 7.5. Fractions were dialyzed against distilled H20, and freeze-dried. Each fraction was then analyzed on a SDS-polyacrylamide gel and those containing the storage proteins were identified. In subsequent preparations, the elution pattern turned out to be very reproducible and the fractions containing the storage proteins were immediately pooled, dialyzed, and freeze-dried. The protein content in each fraction was determined using the Lowry method (20).Polyacrybmide Gel Electrophoresis. To separate proteins in one dimension, the SDS-polyacryLamide gel system (18) was employed, using either 12.5 or 17% slab gels. Gels were stained with amido black or Coomassie blue R-250. Amido black-stained gels were routinely destained and further processed with the silver nitrate staining procedure (24). The isoelectrofocusing cylindrical gels and the second dimension were run as previously described (23, 25) using a pH range 3.5 to 10. The second twodimensional gel system was essentially as already published (22). The first dimension was run on a 1-mm thick slab containing 12.5% acrylamide gel in the absence of2-mercaptoethanol. Each sample was run in duplicate. One strip was stained with Coomassie blue and the other was incubated in sample buffer containing 5% 2-mercaptoethanol to reduce the protein subunits and was placed on the stacking gel of a second dimension 1

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Section: Discussionsupporting

confidence: 83%

“…

(3,11,14,15,19,21In this paper, we characterize radish 12 and 1.7 S proteins and compare them to their rapeseed equivalents. SDS-polyacrylamide gel patterns and amino acid compositions are reported.

…”

mentioning

confidence: 99%

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confidence: 99%

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Characterization of Radish (Raphanus sativus) Storage Proteins

Laroche

Aspart²,

Delseny³

et al. 1984

Plant Physiol.

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“…Aliquots of each fraction were monitored in a y-scintillation counter for content of 1251. Aliquots from every second fraction were analyzed by SDS/polyacrylamide gel electrophoresis in order to determine the sedimentation position of the rapeseed storage proteins napin (1.7 S) and cruciferin (12 S) [9] which served as additional markers.…”

Section: Sedimentation Centrifugation In G Ly Cer Ol Gradientsmentioning

confidence: 99%

Immunological characterization of rapeseed myrosinase

Lenman

Rödin

Josefsson

et al. 1990

European Journal of Biochemistry

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A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75,70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBPSO, respectively). MBP52 and MBP5O lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinasecontaining fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfidelinked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.Many cruciferous plants contain glucosinolates. These are low-molecular-mass compounds consisting of a glucose residue linked via a thioglucoside bond to an amino acid derivative. Some 80 different glucosinolates have been identified, differing mainly in their amino acid portions [l]. The glucosinolates seem to always be accompanied by a group of isoenzymes termed thioglucoside glucohydrolase (EC 3.2.3.1) but mostly referred to by their trivial name myrosinase. This enzyme is capable of catalyzing the hydrolysis of glucosinolates to form glucose, sulfate and, for example, thiocyanates, isothiocyanates, nitriles or epithionitriles. The exact outcome of the reaction is dependent on the substrate as well as on the reaction conditions used. Several of the products have potentially goitrogenic and hepatotoxic effects, which severely restrict the use of rapeseed protein concentrates as animal fodder. Myrosinase has previously been purified from rapeseed and found to be a glycoprotein with a molecular mass of 135 kDa, consisting of two 65-kDa polypeptide chains and containing about 14% carbohydrate [2]. There is evidence for glucosinolates and their degradation products beeing important in determining the specificity of interaction between crucifers and their potential herbivores, pathogens, competitors and symbionts [3]. To understand the physiological significance of the glucosinolate/myrosinase system it is important to study both components. We have produced Abbreviations NaC1/Pi, phosphate-buffered saline, i.e. 8 mM Na2HP04, 1.5 mM KH2P04, 3 mM KC1, 140 mM NaC1, pH 6.8; Tween 20, polyoxyethylene sorbitan monolaurate; ESP, epithiospecifier protein; M75, M70, M65, myrosinases with subunit molecular masses of 75, 70 and 65-kDa, respect...

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“…2). The strong dark band near the top of the gel is the 12S globulin, whereas that of higher mobility near the middle of the gel is the 1.7S protein (Bhatty et al 1968). The Arlo parent appears to contain more protein bands than does the sarson, although the relative concentrations of certain proteins in the seeds (Finlayson et al 1969) from different cultivars may influence the visual recognition of protein bands in the gels.…”

Section: Resultsmentioning

confidence: 99%