Use and Storage of Microsome Products

Microsomes should be stored at -80C. Enzyme activities are stablefor over a year under these storage conditions. Microsome preparations may be frozen and thawed a few times without substantial loss of activity. However, GENTEST recommendsrefreezing small aliquots to minimize freeze-thaw cycles.

To use the microsome product, thaw product rapidly in a 37C waterbath with gentle agitation. Store product on ice after thawing. Product is stable at4C for several hours. Add product to the desired final protein concentration with anappropriate buffer system for your enzyme assay (phosphate or Tris 0.05 to 0.1M work well)and add NADPH or a NADPH generating system. We recommend NADP+ (1 mg/ml), glucose6-phosphate (1 mg/ml) with glucose 6-phosphate dehydrogenase (from yeast) (0.4 u/ml). Theaddition of NADH (0.66 mg/ml) will stimulate some activities via cytochrome b5 or whenusing both NADP+ and NAD+ in a generating system, glucose-6-phosphate dehydrogenase from leuconotucmesenteroides should be substituted for the yeast enzyme. The leuconotucmesenteroides enzyme reduces both NADP+ and NAD+. For the UGT enzymes, NADPH is notneeded, however, the UGT cofactors uridine diphosphate glucuronic acid (1 mM) andmagnesium (5 mM) should be added. Incubations with UGT enzymes should be conducted in Trisbuffer.

Formany cytochrome P450 assays, a 30 minute incubation with 0.5 mg microsomal protein (humanlymphoblast-derived microsomes) or 50 pmole P450 BD Supersomes™ in 1 ml is sufficient toproduce readily detectable quantities of metabolites. Under these conditions, a 1 ml vialor 1 nmole BD Supersomes™ vial of microsomes contains enough material for 20incubations. If higher amounts of metabolites are desired, higher protein/P450concentrations (up to 5 mg/ml protein or 200 pmole/ml P450 BD Supersomes™and/orextension of the incubation times (up to 3 hours) can be used. If sensitive fluorometricor radiometric methods are used to detect metabolites, the protein concentration and/orincubation volume can be decreased; a 1 ml vial or 1 nmole BD Supersomes™ vial ofmicrosomes then contains enough material for about 200 incubations.

When testing a substrate with an enzyme for the first time, we recommend using 0.5 mgmicrosomal protein or 25 to 50 pmole P450 BD Supersomes™ in 0.5 ml buffer/cofactors andincubating for 2 hours at 37C with 50 to 100 uM substrate. Analyze the degree ofmetabolism and then adjust the conditions (protein concentration/substrate concentration/incubation time) depending on the extent of metabolism observed.

Analysis ofcytochrome P450 form-specific metabolism should include a concurrent incubation withcontrol microsomes (Cat. No. 455101 (Old M101A), 455102 (Old M101B),  456201 (Old P201) or 456244 (Old P244)). This provides a controlfor potential metabolism by non-P450 enzymes and for metabolism by the low amount ofnative P450 in the microsomes.

The following specific procedures have been found to improve metabolite production with humanlymphoblast microsomes:

1. Initiate the reaction by adding ice cold microsomesto prewarmed buffer/substrate/cofactors. This procedure often gives higher catalyticactivity than initiating the reaction by adding NADPH to prewarmedmicrosomes/buffer/substrate.
   
2. Use plastic (polypropylene) tubes for theincubation instead of glass. Microsomal protein can adhere to glass and exhibit lowercatalytic activity, particularly, at low protein concentrations. We typically perform a0.5 ml incubation in a 1.5 ml microcentrifuge tube.
   
3. After initial mixing of the microsomes, do notagitate the reaction. Vigorous agitation can denature the protein and agitation is notnecessary for good metabolite production.

Hazard Warning Human LymphoblastMicrosomes
Microsomepreparations are prepared from the human B lymphoblastoid cell line AHH-11 which wasoriginally derived from the RPMI 1788 cell line2. This cell line contains Epstein Barrvirus DNA. The cell lines used to prepare most products contain a vector derived from thepHEBo vector3 which contains DNA sequences derived from the Epstein Barr virus OriPsequences, the E. coli HisD gene4, a human cytochrome P450 cDNA, the plasmidspBR322 and pHSV1065. All products contain some residual sheared cellular DNA. The parentalcell line bearing the OriP-containing vector has been tested and found not to produceinfectious Epstein Barr virus.
References:

1. Mutat. Res. 128: 221-230 (1984).
   
2. ATCC CCL 156
   
3. Mol. Cell. Biol. 5: 410-413 (1985).
4. Mol. Gen. Genet. 203: 382-388 (1986).
5. Nucleic Acids Res. 8: 5949-5964 (1980).

Hazard Warning Insect Cell Microsomes
These products areproduced using baculovirus (Autographa californica) infected insect cells(BTI-TN-5B1-4). This virus is not known to be pathogenic to humans or other mammals.

Safety Recommendations
When using these products, follow good laboratory safety procedures:

• Do not eat, drink or smoke.
  
• Avoid contact with skin or eyes.
  
• Do not inhale aerosols.
  
• Do not pipette by mouth.
  
• Wear suitable protective clothing, gloves and eye protection.
  
• Steam sterilize product or treat product with a 1% solution of sodiumhypochlorite prior to disposal.