Isolation and properties of a thiamine-binding protein from buckwheat seed.
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Three thiamine‐binding proteins of 17‐19 kDa (STBP‐I, II, and III) were purified from sesame seed ( Sesamum indicum L.). Each of the proteins was composed of two subunits of equal molecular mass and each subunit consisted of a large polypeptide and a small polypeptide linked by a disulfide bond(s). They were rich in glutamic acid (or glutamine) and arginine. Their binding activities were optimal at neutral pH. They bound specifically free thiamine but not thiamine phosphates. STBP‐I had higher affinity for thiamine than STBP‐II or STBP‐III. STBP‐II and STBP‐III bound one molecule of thiamine per molecule, and STBP‐I bound 0.5 molecule. The amino acid composition and structure of the STPBs were similar to those of 2S storage proteins.
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Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical procedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10).Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes.In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances.Procedures are described for measuring protein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein. MethodReagents-Reagent A, 2 per cent N&OX in 0.10 N NaOH.Reagent B, 0.5 per cent CuS04.5Hz0 in 1 per cent sodium or potassium tartrabe.Reagent C, alkaline copper solution.Mix 50 ml. of Reagent A with 1 ml. of Reagent B. Discard after 1 day.Reagent D, carbonate-copper solution, is the same as Reagent C except for omission of NaOH.Reagent E, diluted Folin reagent.Titrate Folin-Ciocalteu phenol reagent ((II), Eimer and Amend, Fisher Scientific Company, New York) with NaOH t.o a phenolphthalein end-point.On the basis of this titration dilute the Folin reagent (about 2-fold) to make it 1 N in acid.Working standards may be prepared from human serum diluted IOO-to lOOO-fold (approximately 700 to 70 y per ml.).These in turn may be checked against a standard solution of crystalline bovine albumin (Armour and
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The major storage protein of buckwheat seed is the 13S globulin. Separation of buckwheat seed proteins by sucrose density gradient revealed the existence of an additional new minor storage protein. Analysis of 13S and the new minor class storage proteins by two systems of two-dimensional gel electrophoresis showed that the 13S globulin resembles a structure of legumin-like seed storage proteins, but the new protein appears to be a vicilin-like storage protein. The 57−58 kDa polypeptides, previously described as the unusual subunits of the 13S storage protein, in fact are the subunits of the minor class of buckwheat seed storage proteins. The major and minor classes of storage protein represent about 33 and 6.5% of total seed proteins, respectively. Keywords: Fagopyrum esculentum Moench; buckwheat; seed storage protein
Legumin
Vicilin
Fagopyrum
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Aleurone
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A thiamin-binding protein was isolated from sunflower seeds. Its molecular mass was estimated to be 230 kDa by gel filtration. The protein was suggested to be composed of six subunits, which consisted of polypeptides linked by disulfide bond(s). The protein contained a large amount of glutamine or glutamic acid (19.9 mol%) and asparagine or aspartic acid (11.1 mol%). The levels of tryptophan and valine in the protein were low. These properties of the thiamin-binding protein were similar to those of helianthinin. Optimum pH for the thiamin-binding activity of the protein was 8.0 to 9.0. The thiamin-binding activity was not inhibited by thiamin monophosphate, thiamin pyrophosphate, oxythiamin, or pyrithiamin. These properties of the thiamin-binding protein from sunflower seeds were similar to those from buckwheat seeds, but not to those from rice seeds and sesame seeds.
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Gymnosperm
Bioorganic chemistry
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