Variability of a Double Ring Infiltration Test

Sakai, Kenshi; Upadhyaya, Shrini K.; Sime, M.

doi:10.13031/2013.28723

Cited by 29 publications

(37 citation statements)

References 7 publications

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“…We have been studying the monomer-dimer equilibrium of ␤-lactoglobulin (␤-LG) 1 as a model of protein-protein interaction (1,2). Bovine ␤-lactoglobulin is one of the major whey proteins that is abundant in cow's milk (3)(4)(5), although its function is unknown.…”

mentioning

confidence: 99%

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Sakurai

Goto

2002

Journal of Biological Chemistry

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Bovine ␤-lactoglobulin, a major protein in cow's milk composed of nine ␤-strands (␤A-␤I) and one ␣-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular ␤-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in K a , suggesting that the intermolecular ␤-sheet is essential for dimer formation. By substituting either Asp 33 or Arg 40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K a was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg 40 on one monomer and Asp 33 on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg 33 and Asp 40 of different molecules. These results suggested that protein-protein interactions of bovine ␤-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.

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mentioning

confidence: 99%

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Sakurai

Goto

2002

Journal of Biological Chemistry

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“…Because of this ambiguity and the poor yields of 24-ketocholesterol obtained upon ozonolysis of fucosterol (cf. [la]) an alternative method was used to locate the tritium atom. It has been reported that fucosteryl acetate is isomerised to 3/3-acetoxy-stigmasta-5,24-diene by refluxing with iodine in benzene [17]. When doubly labelled fucosteryl acetate (3H/14C atomic ratio = 3.2315) was isomerised by this method the resulting 3B-acetoxy-stigmasta-5,24-diene had a 3H/I4C atomic ratio of 2.2015.…”

Section: Resultsmentioning

confidence: 97%

“…The method used was based upon that of Sakai [17]. Doubly labelled fucosteryl acetate (2 mg) was refluxed €or 6 h with 3.0 ml of a solution of iodine in benzene (0.9 mg of iodinelml of benzene).…”

Section: Isomerisation Of Fucosteryl Acetate To 3!?-acetoxystigmastamentioning

confidence: 99%

Studies in Phytosterol Biosynthesis: Observations on the Biosynthesis of Fucosterol in the Marine Brown Alga Fucus spiralis

Goad¹,

Goodwin²

1969

European Journal of Biochemistry

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Fucosterol biosynthesised by Fucus spiralis from [2-14C,(4R)-4-3Hl] mevalonic acid has a 3H/14C atomic ratio of 315. Isomerisation of the labelled fucosteryl acetate gave 3/3-acetoxystigmasta-5,24-diene with a 3H/14C atomic ratio of 2/5. This demonstrated that the fucosterol had a 3H atom located a t C-25 and therefore proves that hydrogen (tritium) migration from C-24 to C-25 occurs during alkylation of the phytosterol side chain. Cycloartenol and 24-methylene cycloartanol also isolated from the Fucus spiralis were shown to be radioactively labelled, there was no evidence of radioactive lanosterol. Tritium was shown to be present a t C-3 of the biosynthesised cycloartenol but was absent from C-3 of the fucosterol.Sterol biosynthesis in plants is currently being intensively studied in several laboratories [i J but such work has concentrated mainly on higher plants. Continuing our own investigations on phytosterol biosynthesis we have now examined the marine brown alga Fucus spiralis. The major sterol [2,3] of the brown algae (Phaeophyceae) is fucosterol (I). With I Laminaria saceharina it has been shown previously that the C-24 ethylidene group of fucosterol arises by a double transmethylation reaction from methionine[4] as required by the proposed alkylation mechanism [5, Sj. The present paper provides further information concerning the mechanism of the transmethylation reaction and extends our earlier preUnusual Abbreviations. MVA, mevalonic acid; Tlc, thin ayer chromatography. liminary reports [7,8]. Evidence for the occurrence in Fucus spiralis of cycloartenol (11), the postulated phytosterol precursor 11,9,101, is also present,ed.

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“…5f-1 was cultivated as previously described. 1) Assay of D-AGase. D-AGase activity was assayed by measuring the hydrolysis of N-Ac-D-Glu as previously described.…”

Section: Methodsmentioning

confidence: 99%

Metal-characterization ofN-Acyl-D-glutamate Amidohydrolase fromPseudomonassp. Strain 5f-l

Wakayama

Miura

Oshima

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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Variability of a Double Ring Infiltration Test

Cited by 29 publications

References 7 publications

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Studies in Phytosterol Biosynthesis: Observations on the Biosynthesis of Fucosterol in the Marine Brown Alga Fucus spiralis

Metal-characterization ofN-Acyl-D-glutamate Amidohydrolase fromPseudomonassp. Strain 5f-l

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