RSC Advances
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collected and dialyzed against Tris buffer containing 15% Lysobacter enzymogenes strain OH11. This research was sup-
glycerol.
ported in part by the NIH (R01AI097260), NSFC (31329005), and
a University of Nebraska-Lincoln Redox Biology Center pilot
grant.
In vitro assay of acyl-CoA ligase activity
Enzymatic reactions to generate fatty acyl-CoA were set up using
the following conditions: 10 mM ACL, 0.2 mM CoA, 10 mM ATP,
20 mM fatty acid in DMSO, 5 mM MgCl2, 0.2% (w/v) triton, 200
mM DTT, and 100 mM Tris–HCl pH 7.8. All reactions were
Notes and references
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ꢁ
incubated at 30 C overnight. Samples were analyzed by HPLC
(1220 Innity LC, Agilent Technologies) using a reversed-phase
column (Phenomenex, 4.6 ID ꢂ 150 mm). Water containing 25
mM ammonium acetate and 0.5% acetic acid (solvent A) and
acetonitrile containing 0.5% acetic acid (solvent B) were used as
the mobile phases with a ow rate of 1.0 mL minꢀ1. The HPLC
program was as follows: 10% B in A in the rst 3 min, 10–90% B
in 3–15 min, 90% B in 15–25 min, back to 10% B at 26 min, and
maintained to 30 min.29 The metabolites were detected at 260
nm on a UV detector. MS (Finnigan mat, LCQ) was used to verify
the mass of the reaction products. LC-MS/MS was used to
further conrm the product. An Agilent LC-1200 (Santa Clara,
CA) was connected to a 2.1 ꢂ 100 mm Symmetry ODS column
from Waters (Milford, MA) and a Triple Quadrupole Mass
Spectrometer model 4000 QTrap from ABSciex (Framingham,
MA) operating in either single quadrupole (Q1), enhanced mass
spectrum (EMS), MS/MS or multiple reaction monitoring
(MRM) modes. The samples were injected onto the column and
eluted with 98% mobile phase A (0.1% formic acid in water, J. T.
Baker) to 60% B (0.1% formic acid in acetonitrile, Acros
Organics) over 15 minutes, followed by 5 minutes of 98% B and
5 min of 98% A, all at a ow rate of 0.25 mL minꢀ1
.
Kinetic analysis of acyl-CoA ligase activity
The EnzChek Pyrophosphate Assay Kit (Molecular Probes™)
was used to measure the activities of ACLs. A standard curve (y
¼ 0.0156x + 0.0004, R2 ¼ 0.99603) for the pyrophosphate assay
was generated using pyrophosphate standard as a source of PPi.
To measure the activity of ACLs, the following reagents were
combined in 0.1 mL reaction volumes: 0.4 mM ATP, 0.4 mM
CoA, 0.4 mM free fatty acids in DMSO, 0.2 mM 2-amino-6-
mercapto-7-methyl-purine ribonucleoside (MESG), 1 U purine
nucleoside phosphorylase (PNP), 0.01 U pyrophosphatase, and
various ACLs with nal concentration of 15 mM. Aer incu-
ꢁ
bating at 22 C for 30 minutes, the absorbance at 360 nm was
measured and corrected for background absorbance. The
substrates tested were chemically synthesized (R)-3-hydroxy-7-
methyloctanoic acid (corresponding to the fatty acyl chain in
WAP 8294-A2) and (R)-3-hydroxyloctanoic acid (corresponding
to the fatty acyl chain in WAP 8294-A1). The kinetic parameters
of ACLs were determined by initial velocity experiments using
the coupled assay. These data were globally tted to Michaelis–
Menten equation, yielding Km values for the substrates.
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Acknowledgements
We thank Dr Javier Seravalli and Prof. James Takacs for tech- 24 Y. H. Lee, C. Gallant, H. Guo, Y. Li, C. A. Wang and
nical assistance and Dr Guoliang Qian for providing the
K. G. Morgan, J. Biol. Chem., 2000, 275, 3213.
105758 | RSC Adv., 2015, 5, 105753–105759
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