Chemical Modification of Proteins

Carne, Alex F.

doi:10.1385/0-89603-268-x:311

Cited by 12 publications

(8 citation statements)

References 2 publications

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“…NEM is widely used to probe the functional roles of protein free thiols (Carne, 1994; Chouchani et al, 2011). For example, it inhibits all cysteine peptidases, by alkylating the active site-thiol.…”

Section: Experimental Methodsmentioning

confidence: 99%

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Gonzalez‐Fernandez

Sung

Haswell

et al. 2014

Experimental Eye Research

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Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ~300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP’s free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scanvenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP’s antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.

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Section: Experimental Methodsmentioning

confidence: 99%

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Gonzalez‐Fernandez

Sung

Haswell

et al. 2014

Experimental Eye Research

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“…1997) with the following changes. After washing gel pieces with water and 50% acetonitrile in 200 m m ammonium bicarbonate, pH 8, disulfide bonds were reduced with DTT and cysteines were alkylated with iodoacetic acid as previously described (Carne 1994; Smith 1994). Reagents were removed by several washes in 50% acetonitrile in 200 m m ammonium bicarbonate, pH 8, in the dark.…”

Section: Methodsmentioning

confidence: 99%

“…One microgram of puri®ed radiolabeled 65-kDa protein was digested in solution by treating with 20 ng of TPCK-treated trypsin in 200 mL of 100 mm Tris, pH 8.5 at 378C for 18 h. Proteins in gel pieces cut from Coomassie blue-stained SDS-polyacrylamide gels were digested as previously described (Williams et al 1997) with the following changes. After washing gel pieces with water and 50% acetonitrile in 200 mm ammonium bicarbonate, pH 8, disul®de bonds were reduced with DTT and cysteines were alkylated with iodoacetic acid as previously described (Carne 1994;Smith 1994). Reagents were removed by several washes in 50% acetonitrile in 200 mm ammonium bicarbonate, pH 8, in the dark.…”

Section: Trypsin Digestion Of Isolated Proteinsmentioning

confidence: 99%

Cytosolic O‐glycosylation is abundant in nerve terminals

Cole

Hart

2001

Journal of Neurochemistry

184

126

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Phosphorylation plays a key role in regulating growth cone migration and protein traf®cking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modi®cation: b-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc 1 ). O-GlcNAc modi®cations are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modi®cation that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and

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“…The final concentration of each inactivation agent was 1 mM under appropriate conditions. The listed chemicals were used to modify Trp, carboxyl groups (Asp or Glu), His, hydroxyl groups (Ser or Thr), Cys, disulfide bonds, Tyr, and Arg, respectively . The modified enzyme was incubated at 40 °C for 6 H and enzyme activity was assayed under standard conditions.…”

Section: Methodsmentioning

confidence: 99%

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Huang

Zhang

et al. 2015

Biotech and App Biochem

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Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l-arginase was approximately 33.0 kDa as estimated by SDS-PAGE and 192.0 kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9 mM and 3.8 mM(-1) s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.

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Chemical Modification of Proteins

Cited by 12 publications

References 2 publications

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Cytosolic O‐glycosylation is abundant in nerve terminals

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

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