A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin

Ashok J. Chavan; Hyuntae Kim; Boyd E. Haley; David S. Watt

doi:10.1021/bc00005a007

A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin

Ashok J. Chavan, Hyuntae Kim, Boyd E. Haley, David S. Watt

Molecular and Cellular Biochemistry

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

A GTP photoaffinity probe (¹²⁵I-APTG) was developed that incorporated an [¹²⁵I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the ?-position of GTP through a phos-phonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 µM GTP with an apparent of 5 µM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an ¹²⁵I radiolabel external to the GTP moiety, in contrast to the previously reported [?³²P]-8-N3GTP that possessed an internal ³²P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [¹²⁵I]APTG was used to identify a peptide (ݺ65-79) in the exchangeable GTP-binding domain of the ݭsubunit of tubulin.

Original language	English
Pages (from-to)	337-344
Number of pages	8
Journal	Bioconjugate Chemistry
Volume	1
Issue number	5
DOIs	https://doi.org/10.1021/bc00005a007
State	Published - Sep 1 1990

Funding

Funders	Funder number
National Institute of General Medical Sciences	R01GM035806

ASJC Scopus subject areas

Biotechnology
Bioengineering
Biomedical Engineering
Pharmacology
Pharmaceutical Science
Organic Chemistry

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10.1021/bc00005a007

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title = "A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin",

abstract = "A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the ?-position of GTP through a phos-phonamide linkage. A combination of saturation and GTP protection studies (90\% protection at 25 µM GTP with an apparent of 5 µM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [?32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (ݺ65-79) in the exchangeable GTP-binding domain of the ݭsubunit of tubulin.",

author = "Chavan, \{Ashok J.\} and Hyuntae Kim and Haley, \{Boyd E.\} and Watt, \{David S.\}",

year = "1990",

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doi = "10.1021/bc00005a007",

language = "English",

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pages = "337--344",

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T1 - A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin

AU - Chavan, Ashok J.

AU - Kim, Hyuntae

AU - Haley, Boyd E.

AU - Watt, David S.

PY - 1990/9/1

Y1 - 1990/9/1

N2 - A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the ?-position of GTP through a phos-phonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 µM GTP with an apparent of 5 µM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [?32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (ݺ65-79) in the exchangeable GTP-binding domain of the ݭsubunit of tubulin.

AB - A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the ?-position of GTP through a phos-phonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 µM GTP with an apparent of 5 µM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [?32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (ݺ65-79) in the exchangeable GTP-binding domain of the ݭsubunit of tubulin.

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JF - Bioconjugate Chemistry

IS - 5

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A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin

Abstract

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