- Email: [email protected]
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6000 folds, respectively, of the synaptic soluble fraction. The purification yields of Rab3 GEPI and GEPII are calculated to be both about 4%. It can be estimated that the amounts of Rab3 GEPI and GEPII are both about 0.01% of the total proteins in the synaptic soluble fraction. Rab3 GEPI and GEPII are about 20 and 60% pure, respectively, as estimated by SDS–PAGE followed by protein staining with Coomassie brilliant blue. On the basis of these observations, it can be estimated that the kcat values of Rab3 GEPI and GEPII are both about 40 nmol/min/nmol. Northern and Western blot analyses indicate that Rab3 GEPI and GEPII are expressed in all the rat tissues examined with the highest expression in brain.12 Western blot analysis of the subcellular fractions of rat brain indicates that Rab3 GEPI and GEPII are highly enriched in the synaptic soluble fraction.21 Consistently, immunofluorescence microscopic analysis of primary culture hippocampal neurons from rat embryo indicates that Rab3 GEPI and GEPII are localized at the synaptic release sites.21 Therefore, the preparation of the synaptic soluble fraction is a crical step in the purification of Rab3 GEPI and GEPII. 21
H. Oishi, T. Sasaki, F. Nagano, W. Ikeda, T. Ohya, M. Wada, N. Ide, H. Nakanishi, and Y. Takai, J. Biol. Chem. 273, 34580 (1998).
[8] Purification and Properties of Rab3 GTPase-Activating Protein By FUMIKO NAGANO, TAKUYA SASAKI, and YOSHIMI TAKAI Introduction
The Rab small G protein family consists of nearly 40 members in mammal and 11 members in yeast, and is implicated in intracellular vesicle trafficking.1–6 Like other family members, the Rab family members (Rab proteins) cycle between the GDP-bound inactive and GTP-bound active forms, and the GTP-bound form interacts with their specific effector proteins. The GTP-bound form is converted by the action of the intrinsic 1
C. Nuoffer and W. E. Balch, Annu. Rev. Biochem. 63, 949 (1994). Y. Takai, T. Sasaki, H. Shirataki, and H. Nakanishi, Genes Cells 1, 615 (1996). 3 P. Novick and M. Zerial, Curr. Opin. Cell Biol. 9, 496 (1997). 4 V. M. Olkkonen and H. Stenmark, Int. Rev. Cytol. 176, 1 (1997). 5 F. Schimmo¨ller, I. Simon, and S. R. Pfeffer, J. Biol. Chem. 273, 22161 (1998). 6 O. Martinez and B. Goud, Biochim. Biophys. Acta 1404, 101 (1998). 2
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GTPase activity to the GDP-bound form, which then releases the bound effector proteins. Because GTPase-activating proteins (GAPs) stimulate this reaction, they are assumed to play a crucial role in terminating the functions of the substrate small G proteins. However, it is not yet clear whether GTP hydrolysis is important for Rab proteins to accomplish their functions.2,7 Rab3 GAP, which is the first GAP specific for Rab proteins in mammal, has originally been purified with Rab3A as a substrate from rat brain synaptic soluble fraction.8 This GAP is specifically active on the Rab3 subfamily members (Rab3A, Rab3B, Rab3C, and Rab3D), and prefers the lipid-modified form to the lipid-unmodified form. Of the Rab3 subfamily members, Rab3A and Rab3C are implicated in Ca2⫹-dependent exocytosis, particularly in neurotransmitter release.2 Evidence is accumulating that Rab3A is involved in the docking and/or fusion processes. Although the precise mechanism of Rab3A in the regulation of these processes remains to be clarified, the GTP-bound form might interact with a prefusion complex, thereby preventing fusion.2 The GTPase-deficient mutant of Rab3A inhibits Ca2⫹-dependent exocytosis from PC12 cells and chromaffin cells,9,10 suggesting that Rab3 GAP plays a crucial role in the function of Rab3A. Rab3 GAP shows two bands with molecular weights of about 130,000 (p130) and 150,000 (p150) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).8 The cDNAs of p130 and p150 have been cloned from a human brain cDNA library, and the encoded proteins show no homology to any known protein.8,11 By Northern blot analysis, both p130 and p150 are shown to be ubiquitously expressed. The subcellular fractionation analysis in rat brain indicates that both p130 and p150 are enriched in the synaptic soluble fraction.11,12 p150 is coimmunoprecipitated with p130 from this fraction.11 Recombinant p150 forms a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p130 exhibits the GAP activity toward the Rab3 subfamily members and the catalytic domain is located at the C-terminal region. 7
T. C. Su¨dhof, Neuron 18, 519 (1997). K. Fukui, T. Sasaki, K. Imazumi, Y. Matsuura, H. Nakanishi, and Y. Takai, J. Biol. Chem. 272, 4655 (1997). 9 R. W. Holz, W. H. Brondyk, R. A. Senter, L. Kuizon, and I. G. Macara, J. Biol. Chem. 269, 10229 (1994). 10 L. Johannes, P. M. Lledo, M. Roa, J. D. Vincent, J. P. Henry, and F. Darchen, EMBO J. 13, 2029 (1994). 11 F. Nagano, T. Sasaki, K. Fukui, T. Asakura, K. Imazumi, and Y. Takai, J. Biol. Chem. 273, 24781 (1998). 12 H. Oishi, T. Sasaki, F. Nagano, W. Ikeda, T. Ohya, M. Wada, N. Ide, H. Nakanishi, and Y. Takai, J. Biol. Chem. 273, 34580 (1998). 8
[8]
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In contrast, recombinant p150 neither shows the Rab3 GAP activity nor affects the activity of recombinant p130. These results indicate that Rab3 GAP consists of the catalytic (p130) and noncatalytic (p150) subunits. The role of the noncatalytic subunit of Rab3 GAP is unknown, but Sar1 GAP also consists of the catalytic (Sec23) and noncatalytic (Sec24) subunits, both of which are required for the function of Sar1 in the vesicle budding from the endoplasmic reticulum.13 This chapter describes the assays for the Rab3 GAP activity, the procedures for the purification of native Rab3 GAP from rat brain synaptic soluble fraction, the procedures for the purification of recombinant hexahistidine (His6)-tagged Rab3 GAP from Escherichia coli, and the properties of Rab3 GAP. Materials
Dithiothreitol (DTT), EGTA, Nonidet P-40 (NP-40), and Triton X-100 are purchased from Nacalai Tesque (Kyoto, Japan). EDTA, HEPES, and 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) are purchased from Dojindo Laboratories (Kumamoto, Japan). (p-Amidinophenyl)methanesulfonyl fluoride (APMSF) and isopropyl-웁-D-thiogalactopyranoside (IPTG) are purchased from Wako Pure Chemicals (Osaka, Japan). [웂-32P]GTP (185 TBq/mmol) and [움-32P]GTP (110 TBq/mmol) are obtained from Amersham Pharmacia Biotech (Milwaukee, WI). BA-85 nitrocellulose filters (pore size, 0.45 애m) are purchased from Schleicher & Schuell (Dassel, Germany). All other chemicals are of reagent grade. Lipid-modified and lipid-unmodified Rab3As are purified from the membrane and soluble fractions, respectively, of Spodoptera frugiperda cells (Sf9 cells) infected with the baculovirus carrying the Rab3A cDNA.14 The lipid-modified form of Rab2, Rab3B, Rab3C, Rab3D, Rab5A, and Rab11 are purified from the membrane fraction of Sf9 cells infected with the baculovirus carrying each cDNA in a similar manner. All Rab proteins are dissolved in a buffer containing 20 mM Tris-HCl at pH 8.0, 5 mM MgCl2 , 1 mM EDTA, 1 mM DTT, and 0.6% CHAPS. Expression plasmids, pRSET-p130 and pRSET-p150, are constructed by the following procedures. The 2946-bp fragment containing the complete p130 cDNA coding region with the KpnI sites upstream of the initiator methionine codon and downstream of the termination codon is synthesized by polymerase chain reaction (PCR). This fragment is digested by KpnI and inserted into the KpnI site of pRSETB (Invitrogen BV, Groningen, 13 14
L. Hicke, T. Yoshihisa, and R. Schekman, Mol. Biol. Cell 3, 667 (1992). A. Kikuchi, H. Nakanishi, and Y. Takai, Methods Enzymol. 257, 57 (1995).
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Netherlands). The 4182-bp fragment containing the complete p150 cDNA coding region with the EcoRI sites upstream of the initiator methionine codon and downstream of the termination codon is obtained as follows: The N-terminal fragment (base pairs 1–1151) with the EcoRI site upstream of the initiator methionine codon and with the XbaI site in the coding region is synthesized by PCR. This fragment is digested by EcoRI and XbaI and inserted into the EcoRI and XbaI sites of pBluescript (Stratagene, La Jolla, CA). The C-terminal fragment (base pairs 290–4182) is obtained from screening a human brain cDNA library by hybridization and inserted into the EcoRI site of pBluescript. The fragment (base pairs 1152–4182) is digested by XbaI from the latter plasmid and inserted into the XbaI site of the former plasmid. The ligated fragment is digested by EcoRI and inserted into the EcoRI site of pRSETB. An E. coli strain DE3 is transformed with pRSET-p130 and pRSET-p150. Methods
Assay for Rab3 GAP Activity The Rab3 GAP activity to stimulate the intrinsic GTPase activity of Rab3A is assayed by three methods as follows: (1) standard assay (filter assay), (2) overlay assay, and (3) thin-layer chromatography assay. Standard Assay (Filter Assay). Lipid-modified Rab3A (3 pmol) is incubated at 30⬚ for 10 min in a reaction mixture (10 애l) containing 25 mM Tris-HCl at pH 8.0, 10 mM EDTA, 5 mM MgCl2 , 0.5 mM DTT, 0.3% CHAPS, and 1.5 애M [웂-32P]GTP (1 ⫻ 104 cpm/pmol). The reaction is stopped by adding 2.5 애l of 80 mM MgCl2 . To this mixture (12.5 애l), the sample to be assayed is added in a total volume of 50 애l and further incubated at 30⬚ for 5 min. The reaction is stopped by adding 2 ml of an ice-cold solution containing 20 mM Tris-HCl at pH 7.5, 25 mM MgCl2 , and 100 mM NaCl to the reaction mixture, followed by rapid filtration on BA-85 nitrocellulose filters and washing with the same solution three times. The radioactivity retained on the filter is determined by Cerenkov counting. Overlay Assay. The sample to be assayed is subjected to SDS–PAGE. After semidry Western blotting, the nitrocellulose filter-bound proteins are renatured in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, 0.5 mM MgCl2 , 0.1% Triton X-100, and 5 mM DTT. The filter is incubated at 25⬚ for 10 min with [웂-32P]GTP–Rab3A (3 pmol), which is prepared with the same method as described above, in a buffer containing 25 mM HEPES/NaOH at pH 7.0, 1.25 mM MgCl2 , 0.05% Triton X-100, and 2.5 mM DTT. After the filter is washed with PBS containing 25 mM HEPES/NaOH at pH 7.0, 5 mM MgCl2 , and 0.05% Triton X-100, the
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hydrolysis of [웂-32P]GTP bound to Rab3A is analyzed with Fujix BAS 2000 Imaging Analyzer. Thin-Layer Chromatography Assay. [움-32P]GTP–Rab3A (3 pmol) is made as described above except that [움-32P]GTP is used instead of [웂-32P]GTP. The sample to be assayed is mixed with [움-32P]GTP–Rab3A in a reaction mixture (50 애l) containing 35 mM Tris-HCl at pH 8.0, 12 mM MgCl2 , 2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and 0.06% CHAPS at 30⬚ for 5 min. The mixture is applied to a nitrocellulose filter and then rinsed three times with an ice-cold solution containing 20 mM Tris-HCl at pH 7.5, 25 mM MgCl2 , and 100 mM NaCl. Guanine nucleotides bound to Rab3A are eluted by immersing the filter in a buffer containing 20 mM Tris-HCl at pH 8.0, 20 mM EDTA, 2% SDS, 1 mM GDP, and 1 mM GTP at 65⬚ for 5 min. The released nucleotides are separated on a polyethyleneimine-cellulose thin-layer chromatography plate (Macherey-Nagel, Du¨ren, Germany) with 1 M KH2PO4 at pH 3.4. After developing the thin-layer chromatography plate, the plate is dried, and the GDP and GTP spots are visualized with a short-wave ultraviolet lamp and analyzed with Fujix BAS 2000 Imaging Analyzer. Purification of Native Rab3 GAP The various buffers used in the isolation of native Rab3 GAP are as follows: Buffer A: 20 mM Tris-HCl at pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, and 1 mM DTT Buffer B: 20 mM potassium phosphate at pH 7.5, 0.5 mM EDTA, and 1 mM DTT The steps used in the purification of native Rab3 GAP are as follows: (1) preparation of the synaptic soluble fraction from rat brain, (2) QSepharose FF column chromatography, (3) hydroxyapatite column chromatography, (4) heparin-Sepharose CL-6B column chromatography, and (5) Mono Q PC 1.6/5 column chromatography. All the purification procedures are performed at 0–4⬚. Preparation of Synaptic Soluble Fraction from Rat Brain. The synaptic soluble fraction is prepared from 200 rat brains as described (see the chapter on Rab3 GEP by H. Nakanishi and Y. Takai in this volume15). Q-Sepharose FF Column Chromatography. One-fifth of the synaptic soluble fraction (450 ml, 315 mg of protein) is directly applied to a Q-Sepharose FF column (2.6 ⫻ 23 cm) equilibrated with buffer A. After the column is washed with 600 ml of buffer A, elution is performed with 15
H. Nakanishi and Y. Takai, Methods Enzymol. 329, [7], (2001) (this volume).
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a 600-ml linear gradient of NaCl (0–0.5 M) in buffer A, followed by 120 ml of 0.5 M NaCl in buffer A at a flow rate of 5 ml/min. Fractions of 8 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 62–70. These fractions (72 ml, 36 mg of protein) are collected. The rest of the synaptic soluble fraction is subjected to the same Q-Sepharose column chromatography four times in a similar manner. Hydroxyapatite Column Chromatography. The samples of the five Q-Sepharose column chromatographies are pooled and diluted with 720 ml of buffer B. The sample is applied to a hydroxyapatite column (2.6 ⫻ 6.6 cm) equilibrated with buffer B. After the column is washed with 350 ml of the same buffer, elution is performed with a 500-ml linear gradient of potassium phosphate (20–212 mM) in buffer B, followed by a 150-ml linear gradient (212–500 mM) and 150 ml of 500 mM potassium phosphate in buffer B at a flow rate of 1.25 ml/min. Fractions of 10 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 29–40. These fractions (120 ml, 18 mg of protein) are collected. Heparin-Sepharose CL-6B Column Chromatography. The sample is diluted with 240 ml of buffer A and applied to a heparin-Sepharose CL-6B column (0.5 ⫻ 5 cm) equilibrated with buffer A. After the column is washed with 20 ml of the same buffer, elution is performed with 0.5 M NaCl in buffer A at a flow rate of 0.5 ml/min. Fractions of 1 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 2–6. These fractions (5 ml, 4 mg of protein) are collected. Mono Q PC 1.6/5 Column Chromatography. One-fifth of the sample is diluted with 2 ml of buffer A and applied to a Mono Q PC 1.6/5 column equilibrated with 280 mM NaCl in buffer A. After the column is washed with 2 ml of the same buffer, elution is performed with a 3-ml linear gradient of NaCl (280–500 mM) in buffer A, followed by a 0.5-ml linear gradient of NaCl (0.5–1 M) and 0.5 ml of 1 M NaCl in buffer A at a flow rate of 0.1 ml/min. Fractions of 0.1 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 10 and 11. This GAP activity coincides well with two protein bands with molecular weights of about 130,000 and 150,000 as estimated by SDS–PAGE. These fractions (0.2 ml, 14 애g of protein) are collected. The rest of the heparin-Sepharose sample is subjected to the same Mono Q column chromatography four times in a similar manner. The samples of the five Mono Q column chromatographies are pooled and stored at ⫺80⬚ until use. Expression and Purification of Recombinant Rab3 GAP The buffer used in the isolation of recombinant Rab3 GAP is buffer C: 50 mM sodium phosphate at pH 7.5 and 50 mM NaCl.
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The steps used in the purification of recombinant Rab3 GAP are as follows: (1) cultivation of E. coli and induction of His6-tagged Rab3 GAP, (2) preparation of crude supernatant, and (3) affinity purification of His6tagged Rab3 GAP. Cultivation of E. coli and Induction of His6-Tagged Rab3 GAP. Escherichia coli DE3 transformed with pRSET-p130 or pRSET-p150 is cultured at 37⬚ in 1 liter of LB medium containing 50 애g per ml ampicillin to an OD595 of 0.2. After the addition of IPTG at a final concentration of 1 mM for p130 (0.1 mM for p150), cells are further cultured at 30⬚ for p130 (25⬚ for p150) for 4 hr. All procedures after this step are performed at 0–4⬚. Cells are harvested, suspended in 20 ml of PBS, and washed with 20 ml of PBS. The cell pellet is frozen at ⫺80⬚. Preparation of Crude Supernatant. The cell pellet is quickly thawed at 37⬚ and suspended in 10 ml of buffer C containing 1 mg per ml lysozyme and 400 애M APMSF, and the cell suspension is sonicated at a setting of 60 by an ultrasonic processor (Taitec, Tokyo, Japan) on ice for 10 sec six times at 1-min intervals. The homogenate is centrifuged at 100,000g for 1 hr. The supernatant is used for the affinity purification. Affinity Purification of His6-Tagged Rab3 GAP. One ml of Ni2⫹-NTAagarose beads (Qiagen K.K., Tokyo, Japan) is washed in a batch twice with 4 ml of distilled water; once with 4 ml of a buffer containing 50 mM sodium phosphate at pH 6.3, 300 mM NaCl, and 200 mM imidazole; three times with 4 ml of a buffer containing 20 mM sodium phosphate at pH 7.8 and 0.5 M NaCl. Forty milliliters of the crude supernatant prepared as described above is then incubated with the beads on a rotating wheel for 2 hr. After the incubation, the beads are spun down at 800g for 5 min and washed in a batch once with 10 ml of buffer C and once with 10 ml of buffer C containing 40 mM imidazole. Then, the beads are packed onto a 5-ml disposable syringe, washed with 10 ml of buffer C containing 40 mM imidazole, and eluted with 5 ml of buffer C containing 500 mM imidazole. The eluate is dialyzed with 1 liter of buffer A three times. The purity and protein concentrations are analyzed by SDS–PAGE, followed by protein staining with Coomassie Brilliant Blue. Properties of Rab3 GAP Activity of Recombinant Rab3 GAP. The Rab3 GAP activity is assayed in a manner similar to that described above except that recombinant Rab3 GAP is used instead of native Rab3 GAP. Native Rab3 GAP and recombinant p130 stimulate the GTPase activity of Rab3A in dose-dependent and time-dependent manners, but the specific activity of recombinant p130 is weaker than that of native Rab3 GAP. Recombinant p150 does not show
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Rab3 GAP activity under the conditions where native Rab3 GAP and recombinant p130 show activity. In addition, recombinant p150 does not affect the Rab3 GAP activity of recombinant p130. Substrate Specificity. The Rab3 GAP activity is assayed in a manner similar to that described above except that various Rab proteins are used instead of Rab3A as substrates. Rab proteins are purified from the membrane fraction of Sf9 cells. These Rab proteins are the lipid-modified forms because they are sensitive to Rab GDI. Native Rab3 GAP and recombinant p130 stimulate the GTPase activity of Rab3B, Rab3C, and Rab3D as well as that of Rab3A, but the GAP activity against Rab3B is slightly weaker than that against Rab3A, Rab3C, and Rab3D. These Rab3 GAPs do not catalyze the reaction of other Rab proteins, including Rab2, Rab5A, and Rab11. Requirement of Lipid Modifications of Rab3A. The Rab3 GAP activity is assayed in a manner similar to that described above except that lipidunmodified Rab3A is used instead of lipid-modified Rab3A as a substrate. Native Rab3 GAP or recombinant p130 does not stimulate the GTPase activity of lipid-unmodified Rab3A. Coimmunoprecipitation of p130 and p150 from Rat Brain Synaptic Soluble Fraction. Rat brain synaptic soluble fraction (2.7 mg of protein) is incubated with 26 애g of the anti-p130 or anti-p150 polyclonal antibody bound to 40 애l of protein A-Sepharose in 2 ml of a solution containing 10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 150 mM NaCl, and 1% NP-40. Each immunoprecipitate is subjected to SDS–PAGE, followed by protein staining with Coomassie Brilliant Blue or by immunoblotting with the antip130 and anti-p150 polyclonal antibodies. In these experiments, protein bands corresponding to p130 and p150 are immunoprecipitated with either antibody at a molar ratio of about 1 : 1. These results indicate that p130 and p150 form a complex in the synaptic soluble fraction. Complex Formation of Recombinant p130 and p150. Recombinant p150 (50 pmol) is incubated with recombinant p130 (50 pmol) at 4⬚ for 20 min. Recombinant p130 alone (50 pmol), recombinant p150 alone (50 pmol), or the mixture of recombinant p130 and p150 is subjected to ultracentrifugation using 4.8 ml of a continuous sucrose density gradient (5–40% sucrose in buffer A). Centrifugation is performed at 220,000g for 14 hr. Fractions of 150 애l each are collected. A 20-애l aliquot of each fraction is subjected to SDS–PAGE, followd by protein staining with silver. When purified Rab3 GAP is subjected to the same continuous sucrose density gradient ultracentrifugation, p130 and p150 appear in a single peak with a molecular weight of 300,000. In these experiments, recombinant p130 and p150 appear in a single peak with a molecular weight of about 110,000 and 170,000, respectively. As to the mixture of recombinant p130 and p150, both the
[9]
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proteins mostly shift to the position with a molecular weight of 300,000. The molar ratio of p130 and p150 in the peak fraction is estimated to be about 1 : 1 by SDS–PAGE. These results indicate that p130 interacts directly with p150 and forms a heterodimer. Comments
We have shown the three assay methods for the Rab3 GAP activity. By use of the standard assay, many samples can be assayed for a short period. Therefore, this assay is selected for the purification procedures of native Rab3 GAP. In contrast, the overlay assay is useful for the detection of the protein band showing the Rab3 GAP activity. However, these two assay methods are not accurate and do not distinguish the GAP activity with the nonspecific phosphatase activity or the GTP dissociation activity. Therefore, the GAP activity should be confirmed by thin-layer chromatography assay. Of the purification steps for native Rab3 GAP from rat brain synaptic soluble fraction, Mono Q column chromatography by use of the SMART system (Amersham Pharmacia Biotech) is the most important step to obtain a large amount of purified Rab3 GAP. Small total gel volume and dead volume in this system contribute to low nonspecific adsorption, resulting in the superior recovery. In addition, because there is less dilution in this system, it is possible to achieve sample concentration, which also increases the recovery.
[9] Rabphilin-3: A Target Molecule for Rab3 Small G Proteins By HIROMICHI SHIRATAKI, TAKUYA SASAKI, and YOSHIMI TAKAI Introduction
Rab3A, a member of the Rab small G protein family, is implicated in Ca2⫹-dependent exocytosis, particularly in neurotransmitter release.1,2 Recent analyses of Rab3A-deficient mice have revealed an important insight into Rab3A function. In the hippocampal CA1 region of the mice, synaptic depletion is much faster, although two forms of short-term synaptic 1 2
Y. Takai, T. Sasaki, H. Shirataki, and H. Nakanishi, Genes Cells 1, 615 (1996). L. Jr. Gonzalez and R. H. Scheller, Cell 96, 755 (1999).
METHODS IN ENZYMOLOGY, VOL. 329
Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $35.00
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6000 folds, respectively, of the synaptic soluble fraction. The purification yields of Rab3 GEPI and GEPII are calculated to be both about 4%. It can be estimated that the amounts of Rab3 GEPI and GEPII are both about 0.01% of the total proteins in the synaptic soluble fraction. Rab3 GEPI and GEPII are about 20 and 60% pure, respectively, as estimated by SDS–PAGE followed by protein staining with Coomassie brilliant blue. On the basis of these observations, it can be estimated that the kcat values of Rab3 GEPI and GEPII are both about 40 nmol/min/nmol. Northern and Western blot analyses indicate that Rab3 GEPI and GEPII are expressed in all the rat tissues examined with the highest expression in brain.12 Western blot analysis of the subcellular fractions of rat brain indicates that Rab3 GEPI and GEPII are highly enriched in the synaptic soluble fraction.21 Consistently, immunofluorescence microscopic analysis of primary culture hippocampal neurons from rat embryo indicates that Rab3 GEPI and GEPII are localized at the synaptic release sites.21 Therefore, the preparation of the synaptic soluble fraction is a crical step in the purification of Rab3 GEPI and GEPII. 21
H. Oishi, T. Sasaki, F. Nagano, W. Ikeda, T. Ohya, M. Wada, N. Ide, H. Nakanishi, and Y. Takai, J. Biol. Chem. 273, 34580 (1998).
[8] Purification and Properties of Rab3 GTPase-Activating Protein By FUMIKO NAGANO, TAKUYA SASAKI, and YOSHIMI TAKAI Introduction
The Rab small G protein family consists of nearly 40 members in mammal and 11 members in yeast, and is implicated in intracellular vesicle trafficking.1–6 Like other family members, the Rab family members (Rab proteins) cycle between the GDP-bound inactive and GTP-bound active forms, and the GTP-bound form interacts with their specific effector proteins. The GTP-bound form is converted by the action of the intrinsic 1
C. Nuoffer and W. E. Balch, Annu. Rev. Biochem. 63, 949 (1994). Y. Takai, T. Sasaki, H. Shirataki, and H. Nakanishi, Genes Cells 1, 615 (1996). 3 P. Novick and M. Zerial, Curr. Opin. Cell Biol. 9, 496 (1997). 4 V. M. Olkkonen and H. Stenmark, Int. Rev. Cytol. 176, 1 (1997). 5 F. Schimmo¨ller, I. Simon, and S. R. Pfeffer, J. Biol. Chem. 273, 22161 (1998). 6 O. Martinez and B. Goud, Biochim. Biophys. Acta 1404, 101 (1998). 2
METHODS IN ENZYMOLOGY, VOL. 329
Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $35.00
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GTPase activity to the GDP-bound form, which then releases the bound effector proteins. Because GTPase-activating proteins (GAPs) stimulate this reaction, they are assumed to play a crucial role in terminating the functions of the substrate small G proteins. However, it is not yet clear whether GTP hydrolysis is important for Rab proteins to accomplish their functions.2,7 Rab3 GAP, which is the first GAP specific for Rab proteins in mammal, has originally been purified with Rab3A as a substrate from rat brain synaptic soluble fraction.8 This GAP is specifically active on the Rab3 subfamily members (Rab3A, Rab3B, Rab3C, and Rab3D), and prefers the lipid-modified form to the lipid-unmodified form. Of the Rab3 subfamily members, Rab3A and Rab3C are implicated in Ca2⫹-dependent exocytosis, particularly in neurotransmitter release.2 Evidence is accumulating that Rab3A is involved in the docking and/or fusion processes. Although the precise mechanism of Rab3A in the regulation of these processes remains to be clarified, the GTP-bound form might interact with a prefusion complex, thereby preventing fusion.2 The GTPase-deficient mutant of Rab3A inhibits Ca2⫹-dependent exocytosis from PC12 cells and chromaffin cells,9,10 suggesting that Rab3 GAP plays a crucial role in the function of Rab3A. Rab3 GAP shows two bands with molecular weights of about 130,000 (p130) and 150,000 (p150) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).8 The cDNAs of p130 and p150 have been cloned from a human brain cDNA library, and the encoded proteins show no homology to any known protein.8,11 By Northern blot analysis, both p130 and p150 are shown to be ubiquitously expressed. The subcellular fractionation analysis in rat brain indicates that both p130 and p150 are enriched in the synaptic soluble fraction.11,12 p150 is coimmunoprecipitated with p130 from this fraction.11 Recombinant p150 forms a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p130 exhibits the GAP activity toward the Rab3 subfamily members and the catalytic domain is located at the C-terminal region. 7
T. C. Su¨dhof, Neuron 18, 519 (1997). K. Fukui, T. Sasaki, K. Imazumi, Y. Matsuura, H. Nakanishi, and Y. Takai, J. Biol. Chem. 272, 4655 (1997). 9 R. W. Holz, W. H. Brondyk, R. A. Senter, L. Kuizon, and I. G. Macara, J. Biol. Chem. 269, 10229 (1994). 10 L. Johannes, P. M. Lledo, M. Roa, J. D. Vincent, J. P. Henry, and F. Darchen, EMBO J. 13, 2029 (1994). 11 F. Nagano, T. Sasaki, K. Fukui, T. Asakura, K. Imazumi, and Y. Takai, J. Biol. Chem. 273, 24781 (1998). 12 H. Oishi, T. Sasaki, F. Nagano, W. Ikeda, T. Ohya, M. Wada, N. Ide, H. Nakanishi, and Y. Takai, J. Biol. Chem. 273, 34580 (1998). 8
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In contrast, recombinant p150 neither shows the Rab3 GAP activity nor affects the activity of recombinant p130. These results indicate that Rab3 GAP consists of the catalytic (p130) and noncatalytic (p150) subunits. The role of the noncatalytic subunit of Rab3 GAP is unknown, but Sar1 GAP also consists of the catalytic (Sec23) and noncatalytic (Sec24) subunits, both of which are required for the function of Sar1 in the vesicle budding from the endoplasmic reticulum.13 This chapter describes the assays for the Rab3 GAP activity, the procedures for the purification of native Rab3 GAP from rat brain synaptic soluble fraction, the procedures for the purification of recombinant hexahistidine (His6)-tagged Rab3 GAP from Escherichia coli, and the properties of Rab3 GAP. Materials
Dithiothreitol (DTT), EGTA, Nonidet P-40 (NP-40), and Triton X-100 are purchased from Nacalai Tesque (Kyoto, Japan). EDTA, HEPES, and 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) are purchased from Dojindo Laboratories (Kumamoto, Japan). (p-Amidinophenyl)methanesulfonyl fluoride (APMSF) and isopropyl-웁-D-thiogalactopyranoside (IPTG) are purchased from Wako Pure Chemicals (Osaka, Japan). [웂-32P]GTP (185 TBq/mmol) and [움-32P]GTP (110 TBq/mmol) are obtained from Amersham Pharmacia Biotech (Milwaukee, WI). BA-85 nitrocellulose filters (pore size, 0.45 애m) are purchased from Schleicher & Schuell (Dassel, Germany). All other chemicals are of reagent grade. Lipid-modified and lipid-unmodified Rab3As are purified from the membrane and soluble fractions, respectively, of Spodoptera frugiperda cells (Sf9 cells) infected with the baculovirus carrying the Rab3A cDNA.14 The lipid-modified form of Rab2, Rab3B, Rab3C, Rab3D, Rab5A, and Rab11 are purified from the membrane fraction of Sf9 cells infected with the baculovirus carrying each cDNA in a similar manner. All Rab proteins are dissolved in a buffer containing 20 mM Tris-HCl at pH 8.0, 5 mM MgCl2 , 1 mM EDTA, 1 mM DTT, and 0.6% CHAPS. Expression plasmids, pRSET-p130 and pRSET-p150, are constructed by the following procedures. The 2946-bp fragment containing the complete p130 cDNA coding region with the KpnI sites upstream of the initiator methionine codon and downstream of the termination codon is synthesized by polymerase chain reaction (PCR). This fragment is digested by KpnI and inserted into the KpnI site of pRSETB (Invitrogen BV, Groningen, 13 14
L. Hicke, T. Yoshihisa, and R. Schekman, Mol. Biol. Cell 3, 667 (1992). A. Kikuchi, H. Nakanishi, and Y. Takai, Methods Enzymol. 257, 57 (1995).
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Netherlands). The 4182-bp fragment containing the complete p150 cDNA coding region with the EcoRI sites upstream of the initiator methionine codon and downstream of the termination codon is obtained as follows: The N-terminal fragment (base pairs 1–1151) with the EcoRI site upstream of the initiator methionine codon and with the XbaI site in the coding region is synthesized by PCR. This fragment is digested by EcoRI and XbaI and inserted into the EcoRI and XbaI sites of pBluescript (Stratagene, La Jolla, CA). The C-terminal fragment (base pairs 290–4182) is obtained from screening a human brain cDNA library by hybridization and inserted into the EcoRI site of pBluescript. The fragment (base pairs 1152–4182) is digested by XbaI from the latter plasmid and inserted into the XbaI site of the former plasmid. The ligated fragment is digested by EcoRI and inserted into the EcoRI site of pRSETB. An E. coli strain DE3 is transformed with pRSET-p130 and pRSET-p150. Methods
Assay for Rab3 GAP Activity The Rab3 GAP activity to stimulate the intrinsic GTPase activity of Rab3A is assayed by three methods as follows: (1) standard assay (filter assay), (2) overlay assay, and (3) thin-layer chromatography assay. Standard Assay (Filter Assay). Lipid-modified Rab3A (3 pmol) is incubated at 30⬚ for 10 min in a reaction mixture (10 애l) containing 25 mM Tris-HCl at pH 8.0, 10 mM EDTA, 5 mM MgCl2 , 0.5 mM DTT, 0.3% CHAPS, and 1.5 애M [웂-32P]GTP (1 ⫻ 104 cpm/pmol). The reaction is stopped by adding 2.5 애l of 80 mM MgCl2 . To this mixture (12.5 애l), the sample to be assayed is added in a total volume of 50 애l and further incubated at 30⬚ for 5 min. The reaction is stopped by adding 2 ml of an ice-cold solution containing 20 mM Tris-HCl at pH 7.5, 25 mM MgCl2 , and 100 mM NaCl to the reaction mixture, followed by rapid filtration on BA-85 nitrocellulose filters and washing with the same solution three times. The radioactivity retained on the filter is determined by Cerenkov counting. Overlay Assay. The sample to be assayed is subjected to SDS–PAGE. After semidry Western blotting, the nitrocellulose filter-bound proteins are renatured in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, 0.5 mM MgCl2 , 0.1% Triton X-100, and 5 mM DTT. The filter is incubated at 25⬚ for 10 min with [웂-32P]GTP–Rab3A (3 pmol), which is prepared with the same method as described above, in a buffer containing 25 mM HEPES/NaOH at pH 7.0, 1.25 mM MgCl2 , 0.05% Triton X-100, and 2.5 mM DTT. After the filter is washed with PBS containing 25 mM HEPES/NaOH at pH 7.0, 5 mM MgCl2 , and 0.05% Triton X-100, the
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hydrolysis of [웂-32P]GTP bound to Rab3A is analyzed with Fujix BAS 2000 Imaging Analyzer. Thin-Layer Chromatography Assay. [움-32P]GTP–Rab3A (3 pmol) is made as described above except that [움-32P]GTP is used instead of [웂-32P]GTP. The sample to be assayed is mixed with [움-32P]GTP–Rab3A in a reaction mixture (50 애l) containing 35 mM Tris-HCl at pH 8.0, 12 mM MgCl2 , 2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and 0.06% CHAPS at 30⬚ for 5 min. The mixture is applied to a nitrocellulose filter and then rinsed three times with an ice-cold solution containing 20 mM Tris-HCl at pH 7.5, 25 mM MgCl2 , and 100 mM NaCl. Guanine nucleotides bound to Rab3A are eluted by immersing the filter in a buffer containing 20 mM Tris-HCl at pH 8.0, 20 mM EDTA, 2% SDS, 1 mM GDP, and 1 mM GTP at 65⬚ for 5 min. The released nucleotides are separated on a polyethyleneimine-cellulose thin-layer chromatography plate (Macherey-Nagel, Du¨ren, Germany) with 1 M KH2PO4 at pH 3.4. After developing the thin-layer chromatography plate, the plate is dried, and the GDP and GTP spots are visualized with a short-wave ultraviolet lamp and analyzed with Fujix BAS 2000 Imaging Analyzer. Purification of Native Rab3 GAP The various buffers used in the isolation of native Rab3 GAP are as follows: Buffer A: 20 mM Tris-HCl at pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, and 1 mM DTT Buffer B: 20 mM potassium phosphate at pH 7.5, 0.5 mM EDTA, and 1 mM DTT The steps used in the purification of native Rab3 GAP are as follows: (1) preparation of the synaptic soluble fraction from rat brain, (2) QSepharose FF column chromatography, (3) hydroxyapatite column chromatography, (4) heparin-Sepharose CL-6B column chromatography, and (5) Mono Q PC 1.6/5 column chromatography. All the purification procedures are performed at 0–4⬚. Preparation of Synaptic Soluble Fraction from Rat Brain. The synaptic soluble fraction is prepared from 200 rat brains as described (see the chapter on Rab3 GEP by H. Nakanishi and Y. Takai in this volume15). Q-Sepharose FF Column Chromatography. One-fifth of the synaptic soluble fraction (450 ml, 315 mg of protein) is directly applied to a Q-Sepharose FF column (2.6 ⫻ 23 cm) equilibrated with buffer A. After the column is washed with 600 ml of buffer A, elution is performed with 15
H. Nakanishi and Y. Takai, Methods Enzymol. 329, [7], (2001) (this volume).
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a 600-ml linear gradient of NaCl (0–0.5 M) in buffer A, followed by 120 ml of 0.5 M NaCl in buffer A at a flow rate of 5 ml/min. Fractions of 8 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 62–70. These fractions (72 ml, 36 mg of protein) are collected. The rest of the synaptic soluble fraction is subjected to the same Q-Sepharose column chromatography four times in a similar manner. Hydroxyapatite Column Chromatography. The samples of the five Q-Sepharose column chromatographies are pooled and diluted with 720 ml of buffer B. The sample is applied to a hydroxyapatite column (2.6 ⫻ 6.6 cm) equilibrated with buffer B. After the column is washed with 350 ml of the same buffer, elution is performed with a 500-ml linear gradient of potassium phosphate (20–212 mM) in buffer B, followed by a 150-ml linear gradient (212–500 mM) and 150 ml of 500 mM potassium phosphate in buffer B at a flow rate of 1.25 ml/min. Fractions of 10 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 29–40. These fractions (120 ml, 18 mg of protein) are collected. Heparin-Sepharose CL-6B Column Chromatography. The sample is diluted with 240 ml of buffer A and applied to a heparin-Sepharose CL-6B column (0.5 ⫻ 5 cm) equilibrated with buffer A. After the column is washed with 20 ml of the same buffer, elution is performed with 0.5 M NaCl in buffer A at a flow rate of 0.5 ml/min. Fractions of 1 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 2–6. These fractions (5 ml, 4 mg of protein) are collected. Mono Q PC 1.6/5 Column Chromatography. One-fifth of the sample is diluted with 2 ml of buffer A and applied to a Mono Q PC 1.6/5 column equilibrated with 280 mM NaCl in buffer A. After the column is washed with 2 ml of the same buffer, elution is performed with a 3-ml linear gradient of NaCl (280–500 mM) in buffer A, followed by a 0.5-ml linear gradient of NaCl (0.5–1 M) and 0.5 ml of 1 M NaCl in buffer A at a flow rate of 0.1 ml/min. Fractions of 0.1 ml each are collected. One peak of the Rab3 GAP activity appears in fractions 10 and 11. This GAP activity coincides well with two protein bands with molecular weights of about 130,000 and 150,000 as estimated by SDS–PAGE. These fractions (0.2 ml, 14 애g of protein) are collected. The rest of the heparin-Sepharose sample is subjected to the same Mono Q column chromatography four times in a similar manner. The samples of the five Mono Q column chromatographies are pooled and stored at ⫺80⬚ until use. Expression and Purification of Recombinant Rab3 GAP The buffer used in the isolation of recombinant Rab3 GAP is buffer C: 50 mM sodium phosphate at pH 7.5 and 50 mM NaCl.
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The steps used in the purification of recombinant Rab3 GAP are as follows: (1) cultivation of E. coli and induction of His6-tagged Rab3 GAP, (2) preparation of crude supernatant, and (3) affinity purification of His6tagged Rab3 GAP. Cultivation of E. coli and Induction of His6-Tagged Rab3 GAP. Escherichia coli DE3 transformed with pRSET-p130 or pRSET-p150 is cultured at 37⬚ in 1 liter of LB medium containing 50 애g per ml ampicillin to an OD595 of 0.2. After the addition of IPTG at a final concentration of 1 mM for p130 (0.1 mM for p150), cells are further cultured at 30⬚ for p130 (25⬚ for p150) for 4 hr. All procedures after this step are performed at 0–4⬚. Cells are harvested, suspended in 20 ml of PBS, and washed with 20 ml of PBS. The cell pellet is frozen at ⫺80⬚. Preparation of Crude Supernatant. The cell pellet is quickly thawed at 37⬚ and suspended in 10 ml of buffer C containing 1 mg per ml lysozyme and 400 애M APMSF, and the cell suspension is sonicated at a setting of 60 by an ultrasonic processor (Taitec, Tokyo, Japan) on ice for 10 sec six times at 1-min intervals. The homogenate is centrifuged at 100,000g for 1 hr. The supernatant is used for the affinity purification. Affinity Purification of His6-Tagged Rab3 GAP. One ml of Ni2⫹-NTAagarose beads (Qiagen K.K., Tokyo, Japan) is washed in a batch twice with 4 ml of distilled water; once with 4 ml of a buffer containing 50 mM sodium phosphate at pH 6.3, 300 mM NaCl, and 200 mM imidazole; three times with 4 ml of a buffer containing 20 mM sodium phosphate at pH 7.8 and 0.5 M NaCl. Forty milliliters of the crude supernatant prepared as described above is then incubated with the beads on a rotating wheel for 2 hr. After the incubation, the beads are spun down at 800g for 5 min and washed in a batch once with 10 ml of buffer C and once with 10 ml of buffer C containing 40 mM imidazole. Then, the beads are packed onto a 5-ml disposable syringe, washed with 10 ml of buffer C containing 40 mM imidazole, and eluted with 5 ml of buffer C containing 500 mM imidazole. The eluate is dialyzed with 1 liter of buffer A three times. The purity and protein concentrations are analyzed by SDS–PAGE, followed by protein staining with Coomassie Brilliant Blue. Properties of Rab3 GAP Activity of Recombinant Rab3 GAP. The Rab3 GAP activity is assayed in a manner similar to that described above except that recombinant Rab3 GAP is used instead of native Rab3 GAP. Native Rab3 GAP and recombinant p130 stimulate the GTPase activity of Rab3A in dose-dependent and time-dependent manners, but the specific activity of recombinant p130 is weaker than that of native Rab3 GAP. Recombinant p150 does not show
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Rab3 GAP activity under the conditions where native Rab3 GAP and recombinant p130 show activity. In addition, recombinant p150 does not affect the Rab3 GAP activity of recombinant p130. Substrate Specificity. The Rab3 GAP activity is assayed in a manner similar to that described above except that various Rab proteins are used instead of Rab3A as substrates. Rab proteins are purified from the membrane fraction of Sf9 cells. These Rab proteins are the lipid-modified forms because they are sensitive to Rab GDI. Native Rab3 GAP and recombinant p130 stimulate the GTPase activity of Rab3B, Rab3C, and Rab3D as well as that of Rab3A, but the GAP activity against Rab3B is slightly weaker than that against Rab3A, Rab3C, and Rab3D. These Rab3 GAPs do not catalyze the reaction of other Rab proteins, including Rab2, Rab5A, and Rab11. Requirement of Lipid Modifications of Rab3A. The Rab3 GAP activity is assayed in a manner similar to that described above except that lipidunmodified Rab3A is used instead of lipid-modified Rab3A as a substrate. Native Rab3 GAP or recombinant p130 does not stimulate the GTPase activity of lipid-unmodified Rab3A. Coimmunoprecipitation of p130 and p150 from Rat Brain Synaptic Soluble Fraction. Rat brain synaptic soluble fraction (2.7 mg of protein) is incubated with 26 애g of the anti-p130 or anti-p150 polyclonal antibody bound to 40 애l of protein A-Sepharose in 2 ml of a solution containing 10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 150 mM NaCl, and 1% NP-40. Each immunoprecipitate is subjected to SDS–PAGE, followed by protein staining with Coomassie Brilliant Blue or by immunoblotting with the antip130 and anti-p150 polyclonal antibodies. In these experiments, protein bands corresponding to p130 and p150 are immunoprecipitated with either antibody at a molar ratio of about 1 : 1. These results indicate that p130 and p150 form a complex in the synaptic soluble fraction. Complex Formation of Recombinant p130 and p150. Recombinant p150 (50 pmol) is incubated with recombinant p130 (50 pmol) at 4⬚ for 20 min. Recombinant p130 alone (50 pmol), recombinant p150 alone (50 pmol), or the mixture of recombinant p130 and p150 is subjected to ultracentrifugation using 4.8 ml of a continuous sucrose density gradient (5–40% sucrose in buffer A). Centrifugation is performed at 220,000g for 14 hr. Fractions of 150 애l each are collected. A 20-애l aliquot of each fraction is subjected to SDS–PAGE, followd by protein staining with silver. When purified Rab3 GAP is subjected to the same continuous sucrose density gradient ultracentrifugation, p130 and p150 appear in a single peak with a molecular weight of 300,000. In these experiments, recombinant p130 and p150 appear in a single peak with a molecular weight of about 110,000 and 170,000, respectively. As to the mixture of recombinant p130 and p150, both the
[9]
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proteins mostly shift to the position with a molecular weight of 300,000. The molar ratio of p130 and p150 in the peak fraction is estimated to be about 1 : 1 by SDS–PAGE. These results indicate that p130 interacts directly with p150 and forms a heterodimer. Comments
We have shown the three assay methods for the Rab3 GAP activity. By use of the standard assay, many samples can be assayed for a short period. Therefore, this assay is selected for the purification procedures of native Rab3 GAP. In contrast, the overlay assay is useful for the detection of the protein band showing the Rab3 GAP activity. However, these two assay methods are not accurate and do not distinguish the GAP activity with the nonspecific phosphatase activity or the GTP dissociation activity. Therefore, the GAP activity should be confirmed by thin-layer chromatography assay. Of the purification steps for native Rab3 GAP from rat brain synaptic soluble fraction, Mono Q column chromatography by use of the SMART system (Amersham Pharmacia Biotech) is the most important step to obtain a large amount of purified Rab3 GAP. Small total gel volume and dead volume in this system contribute to low nonspecific adsorption, resulting in the superior recovery. In addition, because there is less dilution in this system, it is possible to achieve sample concentration, which also increases the recovery.
[9] Rabphilin-3: A Target Molecule for Rab3 Small G Proteins By HIROMICHI SHIRATAKI, TAKUYA SASAKI, and YOSHIMI TAKAI Introduction
Rab3A, a member of the Rab small G protein family, is implicated in Ca2⫹-dependent exocytosis, particularly in neurotransmitter release.1,2 Recent analyses of Rab3A-deficient mice have revealed an important insight into Rab3A function. In the hippocampal CA1 region of the mice, synaptic depletion is much faster, although two forms of short-term synaptic 1 2
Y. Takai, T. Sasaki, H. Shirataki, and H. Nakanishi, Genes Cells 1, 615 (1996). L. Jr. Gonzalez and R. H. Scheller, Cell 96, 755 (1999).
METHODS IN ENZYMOLOGY, VOL. 329
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