FAQ

What information do I gain from ATPase assays?

Membrane vesicles containing ABC transporters can be used to measure the ATPase activity of the transporter. Changes in the activity of the transporter in the presence of a test drug indicate that there is an interaction between the test drug and the transporter assayed.

The ATPase assay is designed to detect and indicate the nature of the interaction between the test drug and the transporter. The ATPase assay is conducted in the ATPase activation and ATPase inhibition modes.

In the ATPase activation mode the unstimulated drug transporter is employed. If a test drug significantly stimulates the ATPase activity of the transporter it is possibly a substrate of the transporter.

In the ATPase inhibition mode drug transporter stimulated with an appropriate probe substrate is employed. The test drug’s ability to reduce the transporter’s ATPase activity is measured. The ATPase inhibition mode of the ATPase assay allows a detection of interaction of the drug transporter with test drugs that are inhibitors or slowly transported substrates.

What information do I gain from vesicular transport assays?

Membrane preparations, if they contain inside-out vesicles in adequate amount, can be used to measure the actual transport of the molecules using the vesicular transport assay. If the test drug is a transported substrate of the transporter it will be moved from the buffer into the membrane vesicles. Membrane vesicles can be separated from the rest of the compounds by rapid filtration and the amount of transported substrate can be measured.

The standard vesicular transport assay is an indirect inhibitory assay, performed with cold test articles. This assay provides information on any interaction between the ABC transporter and the test article. The transport of the reporter substrate is measured in the presence of the test article (typically in 7 concentrations) and IC50 is defined as the concentration inhibiting the transport of the probe (reporter) substrate by 50%.

Should radio-labeled form of the investigated compound or adequate analytical methods (LC/MS, HPLC) be available, the vesicular transport assay may be performed in a direct format without the probe (reporter) substrate and may identify substrate nature of the test article (KM and Vmax determination). Direct vesicular transport assay is low throughput assay. It is suitable for low permeability test compounds as high permeability compounds may escape from the vesicles through the lipid bilayer. Standard assays are always recommended before running the direct measurements with the test compound.

What information do I gain from whole cell-based assays (C assay for MDR and MRP1, Hoechst dye extrusion assay for BCRP)?

The C dye and Hoechst dye efflux assays are indirect inhibitory-type whole cell based assays. They provide quantitative information on any interaction between the ABC transporter and the investigated test compound that would affect the transport of the precursor of the fluorescent dye (c AM) or Hoechst 33342 out of living cells by MDR, MRP1 or BCRP. The fluorescent signal is proportional to transporter inhibition, IC50 values are defined.

Whole cell based assays provide information that is relevant to living cells. These methods, together with ATPase and vesicular transport, can give a clear insight into the interaction of the test compound with the transporters assayed.

Are membrane products supplied with the two assays (ATPase or vesicular transport) the same or different in any way?

Membrane preparations always contain some closed membrane vesicles that are in inside-out orientation. In these inside-out vesicle preparations, the transport of substrates across the membrane takes molecules from the buffer into the membrane vesicle. Vesicles sold as “for vesicular assays” are validated for vesicular assays, while vesicles sold as “for ATPase assays” are validated only for ATPase.

Does freezing and thawing ruin membrane vesicles?

Repeated freezing and thawing has moderate effect on the ATPase activity of the transporters. Yet, it can ruin the vesicular structure of the membrane preparation. Thus, it is recommended to store membrane preparations in small aliquots and avoid freeze-thaw cycles.

Which ATP brand is suitable for ATPase assays?

The ATPase assay is very sensitive to the quality of the ATP used. ATP is available from several vendors, but unfortunately some batches do not meet the quality requirements necessary to run the ATPase assay. We have very good experience with ATP purchased from Sigma (see protocol for catalog numbers), so if you are concerned with the quality of ATP we suggest purchasing from this vendor.

Why add glutathione to the assay mix in MRP1 and MRP2 membrane based studies?

The MRP1 and MRP2 transporters are known to transport some small, positively charged or hydrophobic molecules only in the presence of glutathione (co-transport), which is readily available in living cells. Also, in the ATPase assay some drugs only modulate the ATPase activity of the MRP1 and MRP2 transporters in the presence of glutathione. Therefore, we suggest assaying the interaction of the test drugs with the MRP1 and MRP2 transporters in the presence of 2 mM glutathione (added to the assay mix).

What organic solvents can be used in what concentration without affecting the performance of the assay?

Membrane-based assays tolerate the most widespread organic solvents used in the pharmaceutical industry (DMSO, ethanol, methanol) up to 2%. Whole cell-based assays are more sensitive; we suggest keeping the concentration of these solvents under 1%.

What reference substrates do you use?

You can find them for each different assay in the Catalogue.

How many compounds can I test with 1 mg membrane preparations in the classic ATPase assay?

We perform the ATPase assay on 96-well plates, adding 20-40 µg of membrane preparation per well. We run 8 concentrations, 2 duplicates and two types of assays (activation + inhibition) per compound. This way we normally test 1 compound per plate, using up about 84 wells (64 assay + 20 control), using ~2-3.5 mg membrane / plate. For basic screening the number of assays per compound can be decreased according to one’s actual needs.

How many compounds can I test with 1 mg membrane preparations in the vesicular transport assay?

We recommend to use 25-50 µg membrane per well depending on the type of transporter. According to our suggested assay layout 4 wells are needed to define 1 data point (ATP +/ -, duplicates). We test 8 concentrations of a test drug (8x4 wells = 32 wells = 8 data points). This means that minimum 0,8-1,6 mg membrane is needed for one test compound.

Can I perform the vesicular transport assay also with different readouts e.g. HPLC or LC/MS-MS?

Our protocol is optimized for a competition type assay set-up, using a labeled probe (reporter) compound, however, HPLC or LC/MS-MS can also be used.

Why does SOLVO use mostly Sf9 insect cells instead of mammalian cells?

The baculovirus-insect cell system has many advantages over mammalian cells or the membranes prepared from mammalian cells.

  • The baculovirus insect cell system is easy-to-use and gives high expression of the transduced gene..
  • Obviously no other mammalian transporters are present in the insect cells.
  • In addition, the endogenous expression of the insect cell proteins is down-regulated upon baculovirus infection.
  • The MDR transporter expression profile in some cell-lines, particularly the drug-selected ones is somewhat unstable. Additional care must be taken for standardization, validation on a regular basis.

Is it possible to obtain the expression systems (recombinant baculovirus or selected cell lines) from SOLVO Biotechnology for in-house Sf9/mammalian membrane production?

It is not our business policy to sell recombinant baculoviruses containing the transporter cDNA, or the selected mammalian cell lines. We provide well-manufactured and standardized membrane products, equipped with datashe to enable a strict quality control. We believe that a long-term membrane support agreement is more economical for our customers than dedicating their own resources to membrane production. It is our experience that producing standardized, high-expression level membrane preparations has caused problems even in some well prepared labs at large pharmaceutical companies.

What is the difference between the wild type and the mutant (R482G) form of the BCRP transporter?

SOLVO Biotechnology offers two variants of the BCRP/MXR(ABCG2) transporter: the wild type form and one mutant variant. So far only the wild type variant was found in the human population, while the mutant variant was present in some drug-selected cell lines. The substrate specificity of the two forms has also been reported to be different (Ozvegy et al., 2002). Vesicle preparations containing the wild type transporter can be used in human pharmacokinetic studies, while preparations containing the mutant variant are recommended to be utilized in academic research.

Which BCRP membrane preparation should I use in my ATPase and Vesicular Transport assays: Sf9 or mammalian?

The BCRP transporter shows a significantly higher intristic ATPase activity than most ABC transporters. This basal activity has been attributed to activators (BCRP substrates) that are present in the membrane preparations. Depending on the type of membrane preparation, this activating effect can be different.

a. In case of membrane preparations from insect cells, the baseline activity is very high, so most interacting compounds actually inhibit this baseline activity.

b. In case of membranes prepared from mammalian cells (BCRP-M), the baseline activity is lower, and some interacting compounds inhibit, while known transported substrates activate the baseline activity.

c. Recently, the effect of cholesterol on the BCRP ATPase activation was studied, as insect cells harbour much less cholesterol in their membrane than human cells. Cholesterol loading led to a specific and marked improvement of the rate of drug stimulated ATPase and maximal velocity of transport for the substrates studied. Cholesterol loading of BCRP-Sf9 membranes makes their ATPase and transport properties similar to the BCRP-M membranes that contain high levels of endogenous cholesterol. Therefore, cholesterol loaded BCRP overexpressing insect cell membranes (BCRP-HAM-Sf9, HAM means High Activity Membrane) are suitable models to study BCRP function. (Pal et al, JPET 2007.)

How significant is the deviation between lots? Eg. deviation between Km values.

Due to the nature of the expression system, the transporter content of the individual batches varies. Therefore, the vanadate sensitive basal (and maximal) substrate stimulated ATPase activity of the different membrane batches do show some variation (up to 30%). These biochemical parameters are measured during our quality control and are indicated on the data sheet for each individual batch.

However, the relative activity of a test drug compared to the basal and maximal activities is independent on the transporter content. Therefore, Km (EC50) values determined using different membrane batches are comparable.

Is it possible to do vesicular transport assay with MDR membrane?

Yes. SOLVO has introduced the unique MDR/P-gp PREDIVEZ Vesicular Transport Reagent kits that work with N-methyl-quinidine as probe (reporter) substrate. Substrates of MDR are usually very hydrophobic and therefore would escape from the membrane vesicles or stick to the vesicles. SOLVO has managed to develop a vesicular transport assay that is available as a ready-to use kit. For further details of the kit please click here.

Is the rat transporter the rat equivalent of human MDR?

In humans there is one MDR gene, while in rodents there are two genes termed mdr1a and mdr1b. Both rodent proteins show high homology in structure and biochemical characteristics with the human counterpart. Although there is a difference in the tissue localization of the two rodent proteins: Mdr1b is the predominant form in the intestine while Mdr1a appears to play an important role in the BBB.

Is the rat Mrp2 transporter equivalent to the human MRP2 transporter?

The rat Mrp2 transporter shows 72.3% sequence identity and 85.6% sequence similarity with human MRP2. Both transporters are expressed on the canalicular membrane of the liver and are known to be responsible for the transport of some organic molecules and their conjugates to the bile.

What is the possible explanation that some slowly transported substrates inhibit the baseline ATPase activity?

Some ABC transporters show baseline vanadate sensitive ATPase activity even in the absence of any substrates added. This is believed to be due to some endogenous substrate that is present in the membrane preparation. If the compound tested is a substrate with lower turnover, and it competes with the endogenous substrate it might inhibit the baseline vanadate sensitive ATPase activity and causes slight decrease of phosphate liberation. In practice, this phenomenon can often be seen for BCRP and sometimes for MDR.

What kind of rapid filtration apparatus do you recommend for your vesicular transport assays?

Filterplates: [Millipore multiscreen HTS 96 well filter plates with FB filters (Cat. No. MSFBN6B10) or equivalent.

Rapid filtration apparatus: [Multiscreen™ HTS Vacuum Manifold from Millipore (Cat. No MSVMHTS00) or equivalent]

Any filtration device is suitable where filtering and washing of the samples can be accomplished within 2 minutes and the pore size and capacity of the filter is adequate for filtering the vesicles. For pore size anything at or below 0.7 Âľm is suitable. The capacity depends on the area of the filter disc. For 2.5 cm discs “flat” filters (e.g. 0.45 Âľm nitrocellulose) are suitable, but for 96-well plate size discs (around 7 mm in diameter) we found that the speed of filtration is significantly reduced for nitrocellulose filters as the membrane vesicles clog the pores. Therefore, we suggest using glass fiber filters in this case.