반응 #348050

반응 방정식

Leukotriene A4

leukotriene A4

lithium hydroxide

Tris-HCl

prostaglandin B2

→

leukotriene B4

용매

ethanol

ethanol

반응 조건

후처리

1. 1
   기타are disrupted by sonication and cytosolic fraction
2. 2
   기타isolated by centrifugation at 100,000× g for 1 hour
3. 3
   기타for 3-4 minutes
4. 4
   기타at 37° C.
5. 5
   workup.ADDITIONwas added to a final concentration of 75 μM
6. 6
   기타the reaction terminated by the addition of 100 μL of acetonitrile/methanol/acetic acid, 150:50:3 (v/v/v)
7. 7
   기타Protein is precipitated by incubation at -20° C. for 30 minutes
8. 8
   workup.WAITg for 10 minutes
9. 9
   기타A 120 μL aliquot of the supernatant was removed
10. 10
    workup.ADDITION20 μL of 0.35% disodium EDTA is added

실험 절차

Leukotriene A4 hydrolase is determined by a direct enzyme assay of cytosolic fraction of cells treated with antisense oligonucleotides, as described by Ohishi et al. (J. Biol. Chem., 262:10200-10205, 1987). Briefly, HL-60 cells treated with antisense oligonucleotides are disrupted by sonication and cytosolic fraction isolated by centrifugation at 100,000× g for 1 hour. The reaction mixture contains 100 mM Tris-HCl buffer (pH=7.8) and enzyme in a total volume of 50 μL. After preincubating the enzyme for 3-4 minutes at 37° C., leukotriene A4 in ethanol containing lithium hydroxide was added to a final concentration of 75 μM and a final ethanol concentration of 2%. The enzyme was incubated for 1 minute at 37° C. and the reaction terminated by the addition of 100 μL of acetonitrile/methanol/acetic acid, 150:50:3 (v/v/v) containing 0.3 nmol prostaglandin B2 as an internal standard. Protein is precipitated by incubation at -20° C. for 30 minutes followed by centrifugation at 10,000× g for 10 minutes. A 120 μL aliquot of the supernatant was removed and 20 μL of 0.35% disodium EDTA is added. The amount of leukotriene B4 formed in 50 μL aliquot of the resulting solution is determined by reverse phase HPLC using a C18 column. Samples are eluted isocratically with solvent containing acetonitrile/methanol/water/acetic acid (3:1:3:0.006, v/v/v/v, 0.05% disodium EDTA). The absorbance at 270 nm is monitored to quantitate the amount of leukotriene B4 formed.

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