10 research outputs found
Cloning and functional characterization of CYP71D14, a root-specific cytochrome P450 from Petunia hybrida
Microarrays of Phospholipid Bilayers Generated by Inkjet Printing
We report an efficient and reproducible
method to generate a microarray of model biological membranes on a
solid substrate by applying the inkjet printing technology. Although
inkjet printing is currently widely used for industrial fabrication
processes, including biological materials, printing lipid membranes
remains technically challenging due to the hydrophobic nature of droplets
and instability of the lipid bilayer structure against dehydration.
In the present study, we printed lipids onto a glass substrate covered
with a micropatterned membrane of a polymeric phospholipid bilayer.
Polymeric bilayers were formed by the lithographic photopolymerization
of a diacetylene-containing phospholipid, 1,2-bisÂ(10,12-tricosadiynoyl)-<i>sn</i>-glycero-3-phosphocholine (DiynePC). After removal of
nonpolymerized DiynePC with a detergent solution, natural lipid membranes
were incorporated into the polymer-free regions (corrals) by using
an electric-field-based inkjet printing device that can eject subfemtoliter
volume droplets. To avoid rapid dehydration and destabilization, we
preprinted an aqueous solution containing agarose and trehalose onto
the corrals and subsequently printed lipid suspensions (“two-step-printing
method”). After rinsing, stable lipid bilayer membranes were
formed in the corrals. The bilayers were continuous and fluid as confirmed
by fluorescence recovery after photobleaching. We could introduce
multiple bilayer patches having different lipid compositions into
the neighboring corrals. The present results demonstrate that the
combination of a patterned polymeric bilayer and inkjet printing technology
enables efficient, reliable, and scalable generation of the model
membrane microarrays having varied compositions
Photoregulation of Cytochrome P450 Activity by Using Caged Compound
Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP<sup>+</sup>) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP<sup>+</sup> resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 ÎĽm; height, 50 ÎĽm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants
Surface Functionalization of a Polymeric Lipid Bilayer for Coupling a Model Biological Membrane with Molecules, Cells, and Microstructures
We describe a stable and functional model biological
membrane based
on a polymerized lipid bilayer with a chemically modified surface.
A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing
phospholipids, 1,2-bisÂ(10,12-tricosadiynoyl)-<i>sn</i>-glycero-3-phosphocholine
(DiynePC) and 1,2-bisÂ(10,12-tricosadiynoyl)-<i>sn</i>-glycero-3-phosphoethanolamine
(DiynePE). DiynePC formed a stable bilayer structure, whereas the
ethanolamine headgroup of DiynePE enabled functional molecules to
be grafted onto the membrane surface. Copolymerization of DiynePC
and DiynePE resulted in a robust bilayer. Functionalization of the
polymeric bilayer provided a route to a robust and biomimetic surface
that can be linked with biomolecules, cells, and three-dimensional
(3D) microstructures. Biotin and peptides were grafted onto the polymeric
bilayer for attaching streptavidin and cultured mammalian cells by
molecular recognition, respectively. Nonspecific adsorption of proteins
and cells on polymeric bilayers was minimum. DiynePE was also used
to attach a microstructure made of an elastomer (polydimethylsiloxan:
PDMS) onto the membrane, forming a confined aqueous solution between
the two surfaces. The microcompartment enabled us to assay the activity
of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid
bilayers were incorporated together with membrane-bound proteins by
lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid
membrane of functionalized polymeric bilayers and fluid bilayers offers
a novel platform for a wide range of biomedical applications including
biosensor, bioassay, cell culture, and cell-based assay
The Effects of Single Nucleotide Polymorphisms in CYP2A13 on Metabolism of 5-Methoxypsoralen
Microarray of Human P450 with an Integrated Oxygen Sensing Film for High-Throughput Detection of Metabolic Activities
A microarray chip containing human P450 isoforms was
constructed
for the parallel assay of their metabolic activities. The chip had
microwells that contained vertically integrated P450 and oxygen sensing
layers. The oxygen sensing film was made of an organically modified
silica film (ORMOSIL) doped with trisÂ(4,7-diphenyl-1,10-phenanthroline)
ruthenium dichloride (RuÂ(dpp)<sub>3</sub>Cl<sub>2</sub>). Human P450s
(23 types) expressed in <i>E. coli</i> and purified as membrane
fractions were immobilized in agarose matrixes on the oxygen sensing
layer. The activities of P450s were determined by evaluating the fluorescence
intensity enhancement of the oxygen sensor due to the oxygen consumption
by the metabolic reaction. By normalizing the responses with the amounts
of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence
enhancement patterns that were characteristic to the combination of
P450 isoforms and substrate material. The patterns obtained from two
psoralen derivatives resembled each other, whereas a structurally
different substrate (capsaicin) resulted in a distinct pattern. These
results suggest the potential of the microarray to analyze the activities
of diverse P450 isoforms in a high-throughput fashion. Furthermore,
mechanism-based inactivation (MBI) of P450 could be detected by successively
incubating a chip with different substrate solutions and measuring
the residual activities