University of Groningen Development and perspectives of fluorescent receptor assays Janssen, Maria Johanna IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1997 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Janssen, M. J. (1997). Development and perspectives of fluorescent receptor assays: A case study with benzodiazepines Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 05-12-2018
Stability of solubilized benzodiazepine receptors 6.1 Introduction Benzodiazepine and GABA receptors have been solubilized by several researchers for different purposes, such as the characterization of the receptors and the production of antibodies against these receptors. Different detergents can be used for the solubilization of the benzodiazepine receptors, of which sodium deoxycholate provides the maximum number of solubilized GABA and benzodiazepine binding sites [1]. When the results of binding experiments with solubilized receptors have to be compared over more days, it is necessary to have suitable storage conditions for the solubilized benzodiazepine receptors. It has been reported that the benzodiazepine receptor in sodium deoxycholate is not stable for longer periods [1-3]. To obtain a stable solubilized receptor, the detergent has often been exchanged for another detergent, such as Triton X-100 [1,2] or Lubrol PX [3]. The detergent can be exchanged by dialysis or by gel filtration. In this paper, we report our results on the stability of benzodiazepine receptors under different conditions. Stephenson et al. [1] reported that the detergent precipitated out of solution when the receptor was stored at -20 C in the presence of sodium deoxycholate. However, the [ 3 H]flunitrazepam binding affinity was retained after freezing at -20 C in Triton X-100. In our experiments, the influence of the detergent, in which the solubilized receptors were stored, on the binding affinity was studied over a longer period. After solubilization, the sodium deoxycholate from one part of the solubilized receptors was exchanged for Triton X- 100 by dialysis. Both receptor preparations, in sodium deoxycholate and in Triton X-100, were stored under different conditions, at -20 C, -80 C, and at -20 C after lyophilization. The binding capacity for [ 3 H]flunitrazepam was determined at different times during 2 months. We also assessed the influence of different dialysis conditions. Slightly modified version of: M.J. Janssen, K. Ensing and R.A. de Zeeuw, Preparative Biochem., in press. 73
6.2 Materials and methods Chemicals [N-methyl- 3 H]flunitrazepam (82.0 Ci/mmol) was obtained from DuPont NEN (Wilmington, DE, USA). Lorazepam was a gift of Wyeth Laboratoria (Hoofddorp, The Netherlands). Triton X-100 was supplied by Fluka Chemie AG (Buchs, Switzerland) and sodium deoxycholate (>95%), BSA (Fraction V), bovine globulins (Cohn Fraction II,III) and the protease inhibitors were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Polyethylene glycol (PEG) 6000 was obtained from Genfarma (Maarssen, The Netherlands). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). The GF/B glassfibre filters were obtained from Whatman (Maidstone, UK) and the dialysis membranes (Spectra/Por CE) were supplied by Spectrum (Houston, TX, USA). Rialuma, used as scintillation cocktail, was obtained from Lumac (Olen, Belgium). Demineralized water was further purified by an Elgastat Maxima instrument (Elga, High Wycombe, UK) before use in the buffer. Preparation of the solubilized receptors We modified the method for the preparation of membrane-bound receptors, described by Möhler and Okada [4]. Calf brains, obtained from the slaughterhouse and stored at -80 C after discarding the cerebella, were homogenized in 6 volumes (w/v) of ice-cold 0.32 M sucrose in a glass-teflon Potter-Elvehjem homogenizer (RW 20 DZW, Janke & Kunkel, Staufen i.br., Germany) and centrifuged at 1,000 x g for 10 min in a Beckman L8-55 Ultracentrifuge (Beckman Instruments, Mijdrecht, The Netherlands). The supernatant was collected and centrifuged at 100,000 x g for 60 min. The resulting pellet (P 2 ) was resuspended in 50 mm sodium phosphate buffer, ph 7.4, and centrifuged at 100,000 x g for 30 min. This washing step was repeated once. All operations were performed at 4 C. The washed P 2 -pellet was resuspended in 5 volumes (w/v) phosphate buffer, frozen in liquid nitrogen and lyophilized (Hetosicc CD 52-1, Heto, Birkerød, Denmark). The lyophilized P 2 -pellet was stored at -20 C. To solubilize the benzodiazepine receptors [5-7], the lyophilized P 2 -pellet was resuspended with the glass/teflon homogenizer in 50 mm Tris-HCl buffer, ph 7.4, containing 150 mm KCl and the protease inhibitors EDTA (1 mm), benzamidine HCl (1 mm), bacitracin (200 µg/ml) and fresh phenylmethylsulfonyl fluoride (0.3 mm). The concentration of the P 2 -pellet was 8 mg/ml. A 5% (w/v) solution of sodium deoxycholate in demineralized water was added dropwise to the magnetically stirred suspension to a final concentration of 0.5% (w/v). The suspension was stirred for 30 min at 4 C and the solubilized receptors were recovered by centrifuging at 100,000 x g for 15 min and collecting the supernatant. Aliquots of the solubilized receptors were stored immediately at -20 C, at -80 C, or were lyophilized and then 74
Stability of solubilized benzodiazepine receptors stored at -20 C. To another aliquot Triton X-100 was added to a final concentration of 0.2%. After mixing for 30 min at 4 C, one half of this solution was further dialysed, whereas the other half was stored without dialysing in the same way as the solubilized receptors without adding Triton X-100. Dialysis of the solubilized receptors For the dialysis of the solubilized receptors, tube membranes (diameter 10 mm) were used with two molecular weight cuf off's (MWCO's), 10,000 and 300,000, respectively. The solubilized receptors with Triton X-100 (25 ml) were transferred in the membranes and dialysed against about 1000 ml 50 mm Tris-HCl buffer, ph 7.4, containing the inhibitors, 0.2% Triton X-100 and 200 mm KCl. The dialysis was continued either for 2 hours or overnight while stirring the buffer gently at 4 C. After dialysis, the solubilized receptors were stored in the same way as the solubilized receptors without dialysis. Binding assay The stability of the solubilized receptor materials was examined by performing binding assays at different time points during two months. For the binding assay, 20 µl [ 3 H]flunitrazepam solution in 50 mm Tris-HCl buffer, ph 7.4, (4 nm final concentration) were mixed in duplicate with either 20 µl lorazepam solution in Tris-HCl buffer (10 µm final concentration) or 20 µl Tris-HCl buffer. To this mixture 160 µl receptor solution were added, whereby the lyophilized receptors were solubilized in Tris-HCl buffer giving a protein amount of about 200 µg per assay. The total mixture was vortexed and incubated for 45 min at 4 C. The incubation was terminated by the addition of 100 µl Tris-HCl buffer, containing 0.5% (w/v) γ-globulin and 30% (w/v) PEG 6000 to precipitate the solubilized receptors, followed by incubation at 4 C for 13 min [7]. The γ-globulin was added as carrier for the precipitation of the solubilized benzodiazepine receptor [8]. Three ml ice-cold Tris-HCl buffer, containing 7.5% PEG, was added and the mixture was filtered through pre-wetted GF/B filters. The tubes were rinsed twice with 3 ml ice-cold buffer, which was also filtered. The filters were transferred into 6 ml polyethylene tubes and dispersed in 3.5 ml Rialuma. The vials were shaken for 2 hours and counted for 5 min in a Tri-Carb 4000 Packard scintillation counter (Canberra Packard, Groningen, The Netherlands). Protein determination The amount of protein after solubilization was assayed by a modified version of the method developed by Lowry [9], using bovine serum albumin as the standard. Before assaying, the protein was precipitated with trichloroacetic acid, to avoid interference of the Tris-HCl buffer and formation of precipitates caused by the detergent [10]. The protein precipitates were directly dissolved in the so called Lowry reagent "C". 75
6.3 Results and discussion The molecular mass of the bovine benzodiazepine receptor solubilized in sodium deoxycholate has been reported to be 240,000 daltons [11] and 355,000 daltons [1]. It was not clear whether the solubilized receptors would pass the membranes with a MWCO of 300,000. Therefore we selected two membranes with different MWCO, 10,000 and 300,000, respectively. For the membrane with a MWCO of 10,000, it was obvious that the receptor did not pass the membrane. Since sodium deoxycholate has a micellar weight of 1,700-4,200 [12], it is readily dialysable with both membranes. The micellar weight of Triton X-100 is 90,000 [12], so that it will pass the dialysis membranes with some difficultly. After an overnight dialysis, the concentration Triton X-100 inside the membrane was only 38% of the concentration outside the membrane when dialysed with a MWCO of 10,000 and 56% when dialysed with a MWCO of 300,000. To overcome this difficulty, Triton X-100 was added to the solubilized receptors in sodium deoxycholate before dialysis, so only the sodium deoxycholate had to be removed. The influence of the different dialysis conditions is shown in Figure 6.1. The stability of the sodium deoxycholate solubilized benzodiazepine receptors obtained after dialysis, are compared with the solubilized receptors where the deoxycholate was not removed by dialysis after the addition of Triton X-100. The different solubilized receptor materials were stored at - 80 C. Figure 6.1 Stability of sodium deoxycholate-solubilized receptors after different dialysis conditions. Without dialysis, only adding Triton X-100 (λ); MWCO 10,000, during 2 hours (τ); MWCO 300,000, during 2 hours (ν); MWCO 10,000, overnight (σ); MWCO 300,000, overnight (υ). 76
Stability of solubilized benzodiazepine receptors Figure 6.2 Stability of solubilized receptors after different storage conditions, -20 C (υ); -80 C (τ); -20 C after lyophilization (). The filled symbols represent solubilized receptors in sodium deoxycholate and the open symbols represent solubilized receptors where the sodium deoxycholate was exchanged for Triton X-100 by dialysis. (MWCO 10,000, 2 hours dialysis). Binding of [ 3 H]flunitrazepam to the solubilized benzodiazepine receptors was slightly decreased after dialysis overnight, but was not altered thereafter over a period of 2 months. Hence, the decrease in binding to the solubilized receptors, dialysed overnight, can be ascribed to the instability of solubilized receptors at 4 C [10]. This implies that if solubilized receptors have to be dialysed, the dialysis time has to be kept to a minimum. Figure 6.2 shows the influence of the storage conditions on the binding affinity of the solubilized receptors stored in sodium deoxycholate and in Triton X-100 (dialysed with a MWCO of 10,000 during 2 hours). It should be noted that in these experiments the receptors were stored in sodium deoxycholate and not dialysed, and no Triton X-100 was added at all. Our results show that the choice of the detergent is not the critical factor in the stability of the solubilized benzodiazepine receptor, as was described by others [1-3], but that the storage conditions are crucial. The solubilized receptors, both in sodium deoxycholate and Triton X- 100, were not stable when stored at -20 C: After storage for one week, the [ 3 H]flunitrazepam binding was decreased to 20% of the binding capacity of fresh solubilized receptors. On the other hand, both solubilized receptor materials retained their binding ability for [ 3 H]flunitrazepam when stored at -80 C or when lyophilized and then stored at -20 C. However, lyophilized solubilized receptors stored at -20 C may give more variance in the binding capacities over the analysis period of 2 months than the receptors stored at -80 C. 77
In other experiments we got the impression that the quality of the buffer may have an effect on de binding affinity of [ 3 H]flunitrazepam for the receptor. The receptors stored at -80 C were all frozen in the same assay buffer, whereas the lyophilized solubilized receptors were dissolved in Tris-HCl buffer, that was freshly prepared before analyzing. This means that the buffer for dissolving the lyophilized receptors differed each time, which could be the reason for the greater variance in binding affinity. From the above results it can be concluded that there is no need to exchange the detergent sodium deoxycholate for an other detergent, such as Triton X-100. Yet, if the deoxycholatesolubilized benzodiazepine receptors have to be stored for a longer period, the latter must be done at -80 C. Under these conditions solubilized receptors are stable at -80 C for at least 2 months. Alternatively, deoxycholate-solubilized receptors may be lyophilized and then stored at -20 C. References [1] F.A. Stephenson, A.E. Watkins and R.W. Olsen. Physicochemical characterization of detergentsolubilized γ-aminobutyric acid and benzodiazepine receptor proteins from bovine brain, Eur. J. Biochem., 123 (1982) 291-298. [2] T. Asano and N. Ogasawara. Solubilization of the benzodiazepine receptor from rat brain, Life Sci., 26 (1980) 607-613. [3] T. Asano, Y. Yamada and N. Ogasawara. Characterization of the solubilized GABA and benzodiazepine receptors from various regions of bovine brain, J. Neurochem., 40 (1983) 209-214. [4] H. Möhler and T. Okada. Properties of 3 H-diazepam binding to benzodiazepine receptors in rat cerebral cortex, Life Sci., 20 (1977) 2102-2110. [5] E. Sigel, F.A. Stephenson, C. Mamalaki and E.A. Barnard. A γ-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex, J. Biol. Chem., 258 (1983) 6965-6971. [6] E. Sigel and E.A. Barnard. A γ-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex, J. Biol. Chem., 259 (1984) 7219-7223. [7] F.A. Stephenson. Purification and molecular characterization of the γ-aminobutyric acid A receptor, in: Receptor biochemistry, A practical approach, Ed. E.C. Hulme, IRL Press, Oxford (1990) pp. 177-201. [8] P. Cuatrecasas. Isolation of the insulin receptor of liver and fat-cell membranes, Proc. Nat. Acad. Sci. USA, 69 (1972) 318-322. [9] S. Clark. Determination of membrane protein concentration, in: Receptor purification procedures, Eds. J.C. Venter and L.C. Harrison, Alan R. Liss, Inc., New York (1984) pp. 149-161. [10] A. Bensadoun and D. Weinstein. Assay of proteins in the presence of interfering materials, Anal. Biochem., 70 (1976) 241-250. [11] C. Mamalaki, E.A. Barnard and F.A. Stephenson. Molecular size of the γ-aminobutyric acid A receptor purified from mammalian cerebral cortex, J. Neurochem., 52 (1989) 124-134. [12] T. Haga, K. Haga and E.C. Hulme. Solubilization, purification, and molecular characterization of receptors: Principles and strategy, in: Receptor biochemistry, A practical approach, Ed. E.C. Hulme, IRL Press, Oxford (1990) pp. 1-50. 78