A C-terminally elongated form of PHI from porcine intestine

Eriste, Elo; Norberg, Åke; Bonetto, Valentina; Nepomuceno, Diane; Lovenberg, Timothy W.; Sillard, Rannar; Jörnvall, Hans

doi:10.1007/s000180050464

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…Porcine Cox17 was isolated from porcine intestine as a fully oxidized apoprotein. Briefly, porcine intestines were processed by the method described in [15] before size-exclusion chromatography. The Cox17-containing fraction from size-exclusion chromatography was further purified on a preparative cationexchange column at pH 5.2 and thereafter at pH 2.5.…”

Section: Purification Of Porcine Cox17mentioning

confidence: 99%

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Palumaa

Kangur

Voronova

et al. 2004

Biochemical Journal

100

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Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization-MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.

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Section: Purification Of Porcine Cox17mentioning

confidence: 99%

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Palumaa

Kangur

Voronova

et al. 2004

Biochemical Journal

100

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“…A concentrate of thermostable intestinal peptides (CTIP) was prepared from porcine intestines, and an aqueous solution of CTIP was fractionated with ethanol [17], and a fraction precipitating at pH 7.2±0.1 prepared as described [18].…”

Section: Methodsmentioning

confidence: 99%

“…The first HPLC purification step was performed under the same conditions as reported earlier [18], but the purification now followed measurements of cAMP production in SK‐N‐MC cells. An active fraction eluting between 150 and 160 ml was collected and used in the subsequent purification.…”

Section: Methodsmentioning

confidence: 99%

Ser¹³‐phosphorylated PYY from porcine intestine with a potent biological activity

et al. 2001

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We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI-TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser 13 . Phospho-PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC 50 values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho-PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho-PYY potently inhibits forskolin-stimulated cAMP accumulation in SK-N-MC cells with an IC 50 value of 0.5 nM compared to 0.15 nM for non-phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products. ß

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A Novel Form of Neurotensin Post-translationally Modified by Arginylation

Eriste

Norberg

Nepomuceno

et al. 2005

Journal of Biological Chemistry

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A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca 2؉ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the ␥-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca 2؉ mobilization elicited by R-NT is via NTR1.

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A C-terminally elongated form of PHI from porcine intestine

Cited by 3 publications

References 16 publications

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Ser¹³‐phosphorylated PYY from porcine intestine with a potent biological activity

A Novel Form of Neurotensin Post-translationally Modified by Arginylation

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A C-terminally elongated form of PHI from porcine intestine

Cited by 3 publications

References 16 publications

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Ser13‐phosphorylated PYY from porcine intestine with a potent biological activity

A Novel Form of Neurotensin Post-translationally Modified by Arginylation

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Product

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Ser¹³‐phosphorylated PYY from porcine intestine with a potent biological activity