|
Below are the answers to many frequently asked questions submitted by our customers. If there are other questions which you would like to have answered please email them to us at info_gentest@bd.com.
What are microsomes? How are they made?
How stable are GENTEST microsome products?
What are SUPERSOMES™ ?
Do lymphoblast-expressed microsomes contain cytochrome b5?
Which SUPERSOMES™ should I use...plus or minus cytochrome b5?
What is the esterase activity in GENTEST microsome products?
Which CYP2C9 or CYP2D6 allele should I use?
How do I correlate data from cDNA-expressed microsomes to data from human liver microsomes?
Do lymphoblast-expressed microsomes contain endogenous Phase II enzymes?
Which control microsome preparation should I use?
Does total microsomal protein concentration affect enzyme activity?
Does organic solvent concentration affect enzyme activity?
Q:What are microsomes? How are they made?
A: Microsomes are membrane fractions derived from human or animal tissues or cells grown in culture. Most membrane-bound enzymes are associated with microsomes. Microsomal enzymes important in drug metabolism and toxicology are: cytochromes P450, cytochrome P450 reductase, cytochrome b5, flavin-containing monooxygenases (FMOs), glucuronosyl transferases (UGTs), and microsomal epoxide hydrolase.
All GENTEST cDNA-expressed microsome products are isolated from the respective expressing cell line grown in culture using differential centrifugation. Cell homogenate is first centrifuged at 9000xg and the resulting supernatant is then centrifuged at 100,000xg. Microsome products are dilutions of the pellet isolated in the later 100,000xg centrifugation.
Q:How stable are GENTEST microsome products?
A: All GENTEST microsome products are stable for at least two years when stored at -80C. To date, no microsome product has shown a significant loss in enzyme activity when stored at -80C for two years.
Two papers which describe the stability of P450s in human liver microsomes have recently been published. Both papers state that there was no significant decrease in P450 activity after 10 freeze/thaw cycles or storage of the microsomes at -80° C for five years. "Effects of Freezing, Thawing, and Storing Human Liver Microsomes on Cytochrome P450 Activity, Robin E. Pearce et al., (1996) Arch. Biochem. Biophys., 331, 145-169. and "Effects of Freezing, Thawing, and Storage of Human Liver Samples on the Microsomal Contents and Activities of Cytochrome P450 Enzymes", Hiroshi Yamazaki, et al., (1997) Drug Metab. Dispos. 25, 168-174.
Cytochrome P450s are known to lose their activity after thawing. We strongly recommend quickly thawing GENTEST microsomes, placing them on ice, and then freezing the microsomes into smaller aliquots immediately after the initial use. Minimization of both freeze thaw cycles and amount of time the microsomes are above -80C should allow the measurement of consistent P450 activity over time.
Q:What are SUPERSOMES™ ?
A: SUPERSOMES™ are microsomes derived from baculovirus infected insect cells. The baculoviruses used to infect the insect cells have been engineered to express one or more drug metabolizing enzyme cDNAs. SUPERSOMES™ catalyze the same enzymatic reactions with (similar Km values) as lymphoblast-expressed or human liver microsome enzymes. SUPERSOMES™ usually contain much higher enzyme activity than other microsome sources. For example, SUPERSOMES™ contain 5-15X the P450 activity of our current lymphoblast-expressed microsomes.
SUPERSOMES™ microsome products are very useful for in vitro metabolic studies. The high enzymatic activities of SUPERSOMES™ make studies of metabolic turnover easier, especially for those substrates which are turned over slowly. As is the case with all cDNA expression systems, in vitro studies requiring a quantitative comparison of cDNA expressed microsomes to human liver samples must be based on the cytochrome P450 quantity and activity in each microsome source. ("Use of cDNA-Expressed Human Cytochrome P450 Enzymes to Study Potential Drug-Drug Interactions," Crespi, CL and BW Penman, (1997) Adv. Pharmacol. 43, 171-188.)
Q:Do lymphoblast-expressed microsomes contain cytochrome b5?
A: All lymphoblast-expressed microsomes derived from the AHH-1 TK+/- cell line contain approximately 50 pmol/mg of cytochrome b5 (Carcinogenesis (1990) 11, 1293-1300.)
Q:Which SUPERSOMES™ should I use...plus or minus cytochrome b5?
A: Customers have the choice in using SUPERSOMES™ containing cDNA-expressed cytochrome b5. The incorporation of cytochrome b5 into several P450 SUPERSOMES™ products increases the respective cytochrome P450 activity (insect cell microsomes do not contain significant amounts of endogenous cytochrome b5). These b5 containing microsomes are particularly useful for our high throughput P450 inhibition screening method where the turnover of the fluorescent substrate can be increased several fold. However, increases in substrate turnover is substrate specific. For example, there is little change in CYP2C9*1 diclofenac 4’-hydroxylase activity in the presence of b5.
Q:Which CYP3A4 SUPERSOMES™ should I buy, 456202 (Old P202) or 456207 (Old P207)?
A:The recommended use of these two products depends upon your experimental investigation. Catalog number 456202 (Old P202) contains cDNA expressed CYP3A4 (0.5 nmole/0.5 ml), cytochrome P450 oxidoreductase, and cytochrome b5. Catalog number 456207 (Old P207) contains only cDNA expressed CYP3A4 (1.0 nmole/0.5 ml) and cytochrome P450 oxidoreductase. 456207 (Old P207) contains approx. 2x the amount of CYP3A4 (pmole/mg protein) compared to 456202 (Old P202). It is well documented, by ourselves and others, that cytochrome b5 stimulates CYP3A4 activity. Therefore, 456202 (Old P202) contains much more activity than 456207 (Old P207) (10-15 fold increase based on turnover number and pmol product/(mgxmin)).
456202 (Old P202) with its high activity is the best value when you are interested in experiments which determine whether a substrate is metabolized by CYP3A4, the substrate is metabolized slowly, or you wish to generate large amounts of metabolite(s) for further analysis.
456207 (Old P207) is better suited for P450 substrate binding studies since it contains a higher concentration of P450. Some customers also like to use 456207 (Old P207) in experiments which compare CYP3A4 and CYP3A5 (456235 (Old P235)) since the 456235 (Old P235) and 456207 (Old P207) products contain comparable amounts of P450 and neither contains cytochrome b5.
Q:What is the esterase activity in GENTEST microsome products?
A: We have measured esterase activity levels in microsome products using fluorescein diacetate as a substrate. A comparison of control microsome preparations to human liver microsomes (HLM) shows that the activity levels are much lower (~100x) for the cDNA-expressed enzyme microsomes relative to HLM.
Pooled HLM (452161 (Old H161)) 48 nmol/mg/min.
SUPERSOMES™ (456201 (Old P201)) 0.7 nmol/mg/min.
Lymphoblast (455101 (Old M101A)) 0.2 nmol/mg/min.
O:Which CYP2C9 or CYP2D6 allele should I use?
A: Several human cytochromes P450 are polymorphic -- multiple allelic variants are found in the population. Some allelic variants contain amino acid substitutions which significantly reduce enzyme activity. GENTEST uses the established P450 nomenclature to label allelic variants (Pharmacogenetics (1996) 6, 193-201.). The wild type allele is always designated as *1. Therefore, most researchers will prefer to use the *1 allele product unless a study of the effect of allelic variants is desired.
Q:How do I correlate data from cDNA-expressed microsomes to data from human liver microsomes?
A: In vitro studies requiring a quantitative comparison of cDNA expressed microsomes to human liver samples must be based on the cytochrome P450 quantity and activity in each microsome source. The method using relative activity factors (RAF) is summarized in the following papers: "Use of cDNA-Expressed Human Cytochrome P450 Enzymes to Study Potential Drug-Drug Interactions," Crespi, CL and BW Penman, (1997) Adv. Pharmacol. 43, 171-188.
Q:Do lymphoblast-expressed microsomes contain endogenous Phase II enzymes?
A: We have limited information on the Phase II activities of lymphoblast microsomes. For glucuronosyl transferase activity and sulfotransferase activity see: C.L. Crespi, J.D. Altman and M.A. Marletta. "Xenobiotic Metabolism and Mutation in a Human Lymphoblastoid Cell Line", Chem.-Biol. Interactions (1985) 53, 257-272. An additional 12% of the benzo(a)pyrene metabolites produced by the native CYP1A1 present in the parent AHH-1 cell line were released by treatment with beta-glucuronidase. However, no direct conjugation of p-nitrophenol, 3-hydroxybenzo(a)pyrene or 9-hydroxybenzo(a)pyrene was observed using 14C-UDPGA (<0.1 pmole per million cell minutes). This suggests that glucuronosyl transferase(s) may be present in control microsomes (455101 (Old M101A)) but, at a very low level. Treatment with sulfatase did not result in the release of additional benzo(a)pyrene metabolites. Since the sulfotransferases are soluble enzymes there should be no activity in microsomes.
Lymphoblast cells contain an appreciable level (4.7 pmole per million cell minutes) of glutathione S-transferase activity with 1-chloro-2,4-nitrobenzene as a substrate (C.L. Crespi, G.M. Seixas, T.R. Turner, C.G. Ryan and B.W. Penman. "Mutagenicity of 1,2-Dichloroethane and 1,2-Dibromoethane in Two Human Lymphoblastoid Cell Lines", Mutat. Res. (1985) 142, 133-140). However, the distribution of activity between the microsomes and cytosol has not been investigated.
Q:Which control microsome preparation should I use?
A: Control microsomes should be included with experiments using incubations containing cDNA-expressed enzymes in order to elucidate the possibility of metabolism by enzymes endogenous to the host cell line.
Lymphoblast-expressed. Two control microsome products from the same lymphoblast cell line (AHH-1 TK+/-) used to make cDNA-expressed microsomes are available; without expression vectors (455101 (Old M101A)) or with an empty expression vector (455102 (Old M101B)). For lymphoblast-derived microsome products which contain supplemental, cDNA-expressed cytochrome P450 reductase (OR), OR microsomes may be used as a control (455114 (Old M114A)).
SUPERSOMES™ . For SUPERSOMES™ products, two control preparations are available. The insect cell control preparations are microsomes prepared from insect cells infected with wild type baculovirus. Insect control SUPERSOMES™ , in two sizes (456200 (Old P200) and 456201 (Old P201)), are prepared from Hi5 insect cells. These cells are currently used for the expression of all SUPERSOMES™ products except CYP3A7 (456237 (Old P237)). Therefore, all current SUPERSOMES™ products except 456237 (Old P237) should be used in association with insect control SUPERSOMES™ (456200 (Old P200) or 456201 (Old P201)). CYP3A7 is expressed using Sf9 insect cells and should be used in association with Sf9 insect cell control SUPERSOMES™ (456299 (Old P299)). Microsomes which contain cDNA expressed OR and cytochrome b5 (456244 (Old P244)) are also available for use as a control.
Q:Does total microsomal protein concentration affect enzyme activity?
A: Differences in total protein concentration can affect the free concentration of substrate and hence cytochrome P450 enzyme kinetics. The available substrate concentration may be significantly lower than initially assumed for those substrates which have a large potential to bind to protein or microsomal membranes. (Obach et al. (1997) J. Pharmacol. Exp. Ther. 283, 46-58 and RS Obach (1997) Drug Metab. Dispos. 25, 1359-69) Total microsomal protein concentration can be controlled and normalized in each incubation by the addition of control microsomes.
Q:Does organic solvent concentration affect enzyme activity?
A: Organic solvents used to dissolve substrates can inhibit cytochrome P450 enzyme activity (Chauret et al. (1998) Drug Metab. Dispos. 26, 1-4 and Busby et al., (1999) Drug Metab. Dispos. 27, 246-249). Low concentrations of organic solvents should be used whenever possible. The use of acetonitrile or methanol is recommended since these solvents have the lowest P450 inhibition potential of several common organic solvents. Organic solvent concentration should be controlled and normalized in each incubation of an experiment.
|
