Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase)

Anil K. Tyagi, David A. Cooney, Marianna Bledsoe, Hiremagalur N. Jayaram

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, l-[4.14C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted l-[4-14C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [14C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADPMgCl2 or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [14C]SAICAR, l-[4-14C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.

Original languageEnglish (US)
Pages (from-to)123-132
Number of pages10
JournalJournal of Biochemical and Biophysical Methods
Volume2
Issue number3
DOIs
StatePublished - 1980
Externally publishedYes

Fingerprint

Ligases
Liver
Aspartic Acid
Scintillation
Spectrometry
Spectrum Analysis
Buffers
Enzymes
Tromethamine
Decarboxylation
Viscera
Biosynthesis
Columbidae
Acetone
Powders
Chickens
Assays
Carbon
phosphoribosylaminoimidazole-succinocarboxamide synthetase
SAICAR

Keywords

  • 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase
  • purine biosynthesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase). / Tyagi, Anil K.; Cooney, David A.; Bledsoe, Marianna; Jayaram, Hiremagalur N.

In: Journal of Biochemical and Biophysical Methods, Vol. 2, No. 3, 1980, p. 123-132.

Research output: Contribution to journalArticle

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abstract = "Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, l-[4.14C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted l-[4-14C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [14C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADPMgCl2 or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [14C]SAICAR, l-[4-14C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40{\%} KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.",
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