Characterization of THLE-Cytochrome P450 (P450) Cell Lines: Gene Expression Background and Relationship to P450-Enzyme Activity

2012 ◽  
Vol 40 (11) ◽  
pp. 2054-2058 ◽  
Author(s):  
Yalda Soltanpour ◽  
Constanze Hilgendorf ◽  
Marie M. Ahlström ◽  
Alison J. Foster ◽  
J. Gerry Kenna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Cell Lines ◽  
P450 Enzyme

scholarly journals Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer–micelle association

2006 ◽  
Vol 395 (3) ◽  
pp. 641-652 ◽  
Author(s):  
Richard K. Hughes ◽  
Eric J. Belfield ◽  
Mylrajan Muthusamay ◽  
Anuja Khan ◽  
Arthur Rowe ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Medicago Truncatula ◽  
Water Soluble ◽  
Hydroperoxide Lyase ◽  
Cytochrome P450 Enzyme ◽  
Cytochrome P450 Enzymes ◽  
P450 Enzyme ◽  
Detergent Micelles

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/Km of up to 1.6×108 M−1·s−1 is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C–C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

BIOM-45. CHARACTERIZATION OF EVs RELEASED FROM 5-ALA DOSED GLIAL AND EXTRA-AXIAL TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi21-vi21
Author(s):  
Tiffaney Hsia ◽  
Anudeep Yekula ◽  
Bob Carter ◽  
Leonora Balaj
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Cell Lines ◽  
Fluorescence Analysis ◽  
Illumina Miseq ◽  
Biological Regulation ◽  
Guided Surgery ◽  
Antibody Staining ◽  
Fluorescence Guided Surgery

Abstract INTRODUCTION The administration of 5-Aminolevunlinic acid (5-ALA) in the context of fluorescence-guided surgery is contingent on the highly selective accumulation of PpIX in tumor cells. PpIX is a precursor in the heme pathway that naturally exhibits fluorescent and phototoxic properties upon excitation. We have previously reported evidence of PpIX fluorescence in tumor-specific extracellular vesicles (EVs). Here, we explore the implications of exogenous 5-ALA on cellular function, EV characteristics, and gene expression. METHODS We have characterized a range of glioblastoma, meningioma, and healthy cell lines (Gli36 EGFRvIII, Gli36 EGFR WT, U87, CH-157, IOMM-Lee, and HBMVEC). At equal confluency, cells were dosed with 0.8 mM 5-ALA or mock dosed. Media was collected and processed to eliminate debris after 24 hours. Analysis of cells and EVs was conducted using Amnis ImageStream (IFC) and Nanoparticle Tracking Analysis. EV subpopulations were analyzed with IFC following antibody staining. RNA was extracted using Qiagen exoRNeasy and RNeasy. Libraries were prepared via Qiagen UPX 3’ Transcriptome kit and sequenced using Illumina MiSeq. Data analysis was carried out via GeneGlobe, CLC workbench, and MATLAB. RESULTS Following 5-ALA dosing, all tumor cells and their derived EVs exhibit significant levels of fluorescence. Analysis of EV subpopulations demonstrated a general decrease in tetraspanin-positive EVs following 5-ALA dosing. At 24 hours, Gli36 cell lines exhibited increased EV release post 5-ALA whereas U87 and meningioma lines resulted in a decreased EV release rate. This clustering is also reflected in EV size distributions and in cellular and EV differential gene expression analysis. Gene ontology analysis of common genes from mock and dosed EVs demonstrated high counts of genes controlling biological regulation as well as cellular and metabolic processes. CONCLUSION Collection and characterization of cancer specific EVs may be advantageous to liquid biopsy development.

Assessing orally bioavailable commercial silver nanoparticle product on human cytochrome P450 enzyme activity

2014 ◽  
Vol 9 (4) ◽  
pp. 474-481 ◽  
Author(s):  
Mark A. Munger ◽  
Greg Hadlock ◽  
Greg Stoddard ◽  
Matthew H. Slawson ◽  
Diana G. Wilkins ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Silver Nanoparticle ◽  
Cytochrome P450 Enzyme ◽  
Human Cytochrome ◽  
P450 Enzyme ◽  
Orally Bioavailable ◽  
Human Cytochrome P450 Enzyme

Effect of Age and Postoperative Time on Cytochrome P450 Enzyme Activity Following Liver Transplantation

2005 ◽  
Vol 45 (6) ◽  
pp. 666-673 ◽  
Author(s):  
Shanshan Liu ◽  
Reginald F. Frye ◽  
Robert A. Branch ◽  
Raman Venkataramanan ◽  
John J. Fung ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Cytochrome P450 Enzyme ◽  
P450 Enzyme ◽  
Postoperative Time ◽  
Effect Of Age
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