Processos de produção da proteína heteróloga m&lt;I&gt;Cherry&lt;/i&gt; por&lt;i&gt; Chlamydomonas reinhardtii&lt;/i&gt;

Arias, D. I.; Andrés, César

doi:10.11606/t.9.2017.tde-06122017-102518

Cited by 12 publications

(15 citation statements)

References 49 publications

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“…The dependence on pH of the binding of oestrone and oestrone sulphate by ligandin was investigated by carrying out experiments in 0.2 M NaCl/5 mM sodium acetate, 0.2 M NaCl/5 mM sodium phosphate and 0.2 M NaC1/5 mM Tris buffers (pH range 4- 10) at a protein concentration of 10 pM and ligand concentrations of 10 nM. The effects of ionic strength on the binding of these two ligands were studied in 0.025 M potassium phosphate buffer, pH 7.0, containing varying concentrations of KCl (0-1.4 M), again at protein and ligand concentrations of 10 pM and 10 nM respectively.…”

Section: Methodsmentioning

confidence: 99%

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The Non‐Covalent Binding of Small Molecules by Ligandin

Tipping

Ketterer

Christodoulides

et al. 1976

European Journal of Biochemistry

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1. Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 x lo', 6.6 x lo5 and 2.6 x lo5 l/mol respectively at pH 7.0,10.16 M, 4 "C. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2-hydroxyoestradiol-glutathione, oestrone glucuronide, N-methyl-4-aminoazobenzene-glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were in the range 1 x lo4-1 x lo6 l/mol.2. Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities, complete binding isotherms could not be obtained. These included several steroids (but not cortisol), 20-methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1 : 1 competition at a single binding site.3. In general the conjugation of non-polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small ( % 15 %, in terms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the observations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions.4. As well as non-polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an association constant of 1 x lo5 l/mol. Glutathione was able to cause the dissociation of the ligandin-oestrone sulphate complex, but this efTect was not explicable in terms of simple 1 : 1 competition.5. Both oestrone and oestrone sulphate were bound most strongly at pH 6-7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum.6. The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N-dimethyl-4-aminoazobenzene are similar in magnitude to those of serum albumin and aminoazodye-binding protein A

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Section: Methodsmentioning

confidence: 99%

“…Because of its non-covalent binding properties it has been postulated that ligandin is a major determinant of the net flux of its ligands from plasma to liver [10,11] and from liver to bile [l]. The protein may also be important in the regulation of the amounts of its ligands available for intracellular metabolism or physiological action [ll].…”

mentioning

confidence: 99%

The Non‐Covalent Binding of Small Molecules by Ligandin

Tipping

Ketterer

Christodoulides

et al. 1976

European Journal of Biochemistry

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“…This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1,2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3)(4)(5)(6)(7).…”

mentioning

confidence: 89%

The Identity of Glutathione S -Transferase B with Ligandin, a Major Binding Protein of Liver

Habig

Pabst

Fleischner

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

470

186

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Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody pioduced against ligandin, bear entirely sinlilar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was showh to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.ligandin is a cytoplasmic protein found in abundance in the liver of rat, man, and other species. This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1, 2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3-7). Fractionation of the protein on the basis of any one of its binding activities has resulted in apparently identical, highly purified preparations (7-9); Thus, the term, ligandin (2), is synonymous with that of azo-dye carcinogenbinding protein (8), corticosteroid binding I protein (9), and Y protein (7)$, §.The glutathione S-transferases (EC 2.5.1.18) from rat liver (iO-i3) have sevieral physical properties in common with rat liver ligandin (2). When crude liver extracts were subjected to filtration on Sephadex G-75, the fraction containing ligandin also served as a source of enzymatic activity for the conjugation of glutathione (GSH) with 1,2-dichloro-4-nitrobenzene (14). However, subsequent fractionation resulted in removal of alriost all GSH transferase activity with this substrate despite virtually complete recovery of ligandin (t, 15).Since four of the GSH transferases of rat liver (transferases A, B; C, and E) have been purified to homogeneity (11-13), (w/v) and the precipitate was removed by centrifugation after an additional 4 hr at 4°. The superratant fluid was used directly for enzyme assays with 1,2-ehloro-4-nitrobenzene. The precipitate was washed twice with phosphate-buffered saline at pH 7.4, containing 2%o polyethylene glycol. The residue was dissolved in 0.5 M NaOH, and absorbance at 280 nm was determined.

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“…woman sampled three times did not exhibit any inhibitory effect or high free fatty acid content. Bilirubin, although difficult to utilize in the experimental conditions chosen (3,5,(23)(24)(25), behaves undoubtedly similar to BSP, at least in regard to the overall competition of fatty acids observed in vitro. A study utiliring radioactive bilirubin is underway.…”

Section: Fig 2 Binding Of Sodium Hromosulfophthalein ( B S P ) To Tmentioning

confidence: 99%

Breast Milk Jaundice: In Vitro Inhibition of Rat Liver Bilirubin-Uridine Diphosphate Glucuronyltransferase Activity and Z Protein-Bromosulfophthalein Binding by Human Breast Milk

et al. 1976

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Processos de produção da proteína heteróloga m<I>Cherry</i> por<i> Chlamydomonas reinhardtii</i>

Cited by 12 publications

References 49 publications

The Non‐Covalent Binding of Small Molecules by Ligandin

The Non‐Covalent Binding of Small Molecules by Ligandin

The Identity of Glutathione S -Transferase B with Ligandin, a Major Binding Protein of Liver

Breast Milk Jaundice: In Vitro Inhibition of Rat Liver Bilirubin-Uridine Diphosphate Glucuronyltransferase Activity and Z Protein-Bromosulfophthalein Binding by Human Breast Milk

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