A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity

Luisa Rios-Avila, Sara A. Prince, Subhashinee S K Wijeratne, Janos Zempleni

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila. Methods: HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy. Results: Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1 ± 1.5 μmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08. nmol/l. Jurkat cells contained 0.14 ± 0.02. U of HCS activity [μmol of biotinylated p67 formed/(nmol/l HCS. h)] in 400 μg of total protein. Conclusions: We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.

Original languageEnglish (US)
Pages (from-to)735-739
Number of pages5
JournalClinica Chimica Acta
Volume412
Issue number9-10
DOIs
StatePublished - Apr 11 2011

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Assays
Throughput
Biotinylation
Histones
Holocarboxylase Synthetase Deficiency
holocarboxylase synthetases
Jurkat Cells
Streptavidin
Genomic Instability
Mammals
Biotin
Cell Extracts
Catalysis
Drosophila
Metabolism
Gene expression
Spectrum Analysis
Fatty Acids
Infrared spectroscopy
Genes

Keywords

  • Assay
  • Biotin
  • Holocarboxylase synthetase
  • Human
  • Recombinant

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity. / Rios-Avila, Luisa; Prince, Sara A.; Wijeratne, Subhashinee S K; Zempleni, Janos.

In: Clinica Chimica Acta, Vol. 412, No. 9-10, 11.04.2011, p. 735-739.

Research output: Contribution to journalArticle

Rios-Avila, Luisa ; Prince, Sara A. ; Wijeratne, Subhashinee S K ; Zempleni, Janos. / A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity. In: Clinica Chimica Acta. 2011 ; Vol. 412, No. 9-10. pp. 735-739.
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N2 - Background: Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila. Methods: HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy. Results: Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1 ± 1.5 μmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08. nmol/l. Jurkat cells contained 0.14 ± 0.02. U of HCS activity [μmol of biotinylated p67 formed/(nmol/l HCS. h)] in 400 μg of total protein. Conclusions: We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.

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