title: A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity.
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 HCSh)] 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.
authors.affiliation: Department of Nutrition & Health Sciences, University of Nebraska-Lincoln, NE 68583-0806, USA.
authors.forename: Luisa
authors.initials: L
authors.lastname: Rios-Avila
authors.forename: Sara A
authors.initials: SA
authors.lastname: Prince
authors.forename: Subhashinee S K
authors.initials: SS
authors.lastname: Wijeratne
authors.forename: Janos
authors.initials: J
authors.lastname: Zempleni
mesh_terms: D002138:Calibration; D019731:Carbon-Nitrogen Ligases / Q000302:isolation & purification / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D010455:Peptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases