Purification and characterization of a low Mr GTP-binding protein, ram p25, expressed by baculovirus expression system

162

Biochimica et Biophysica Acta, 1159 (1992) 162-168 £, 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34299

Purification and characterization of a low M r GTP-binding protein, r a m p25, expressed by baculovirus expression system Takeshi Suzuki a, Koh-ichi Nagata a, Yoshiharu Matsuura b, Yukio O k a n o ~l and Yoshinori Nozawa " " Department of Biochemisto', Gifu Unicersity School of Medicine, Tsukasamachi, Gila (Japan)

and b Department of Veterinary Science, National Institute of Health, Tokyo (Japan) (Received 6 April 1992)

Key words: ram p25; Expression system: Protein purification: GTP binding protein: (Baculovirus)

The r a m gene was isolated from rat megakaryocyte cDNA library with an oligonucleotide probe which is specific for a low M r GTP-binding proteins c25KG purified from human platelets. Its gene product ( r a m p25) is a monomeric 25-kDa guanine nucleotide-binding protein. The protein was expressed by using baculovirus transfer vector, pAcYM1, which allowed the production at a high level of soluble recombinant r a m p25 in S p o d o p t e r a f r u g i p e r d a (Sf9) cells under the control of polyhedrin promoter. The expressed protein in cytosol of Sf9 cells was purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. The purified r a m p25 bound approx. 0.8 _+0.02 mol of guanosine 5'-Ool-thiotriphosphate (GTPTS)/mol of protein with a K d value of 340 + 4.91 nM in a reaction mixture containing 10 p.M of free magnesium ions. In the presence of 5 mM Mg 2+, [3H]GDP was dissociated from r a m p25 at the rate of 0.015 _+0.0010 min i and the dissociation was greatly enhanced by addition of 250 mM (NH4)2SO 4. The rate of [7-3zp]GTP-hydrolysis for r a m p25 was 0.010 _+0.0012 min 1. Thus, it was indicated that the GTP-hydrolysis reaction is a rate-limiting step in the guanine nucleotide turnover of r a m p25. r a m p25 shares 23 and 80% amino-acid homology with the H a - r a s p21 and c25KG protein. respectively, and is similar to them in GTPTS binding activity in a time- and dose-dependent manner. But it differs from ras p21 in the rate-limiting step of the guanine nucleotide turnover.

Introduction There is a superfamily of structurally homologous monomeric GTP-binding proteins ( M r 20000-30000) in mammalian cells [1-3]. These low-M r GTP-binding proteins exhibit 3 0 - 5 0 % homology with r a s p21 [4] and are considered to be involved in signal transduction across the plasma membrane, control of differentiation and proliferation, translocation of nascent proteins into the endoplasmic reticulum, vesicular traffic within the cell and the regulation of superoxide production [5-10]. r a m p25 belongs to this superfamily whose gene has been identified in rat megakaryocyte c D N A library with a synthetic oligonucleotide probe corresponding to an 8-amino-acid sequence, specific for a low M r GTP-binding protein c25KG purified from human platelets [11]. This protein is composed of 221 amino acids with a calculated M r of 25 068 and shares 23 and 80% amino-acid homology with the H a - r a s p21 and

Correspondence to: Y. Nozawa, Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan.

c25KG protein, respectively [12]. Its consensus aminoacid sequences are in GTP-binding and GTPase domains which have been reported for other Iow-M r GTP-binding proteins [3,4]. r a m p25 is thought to be a m e m b e r of the r a b family, on the basis of the aminoacid sequence homology [12]. The function(s) of r a m p25 is unknown at present, but it shares 40% aminoacid identity and 60% homology with yeast S E C 4 protein [13], suggesting that r a m p25 might play some role in the secretory pathway. For further biochemical characterization of r a m p25, we employed a baculovirus expression system to obtain sufficient amounts of r a m p25. An alternative higher eukaryotic expression procedure is the baculovirus expression system which has been used successfully for the production of authentic post-translationally modified and processed recombinant proteins [14-16]. In this system, the inserted gene is placed under control of the polyhedrin promoter of the baculovirus A u t o g r a p h a c a l i f o r n i c a . Recombinant virus obtained by this procedure is used to infect cultured insect cells and, in some instances, is capable of producing high amounts of recombinant protein. One of the major advantages

163 of this invertebrate virus expression vector over bacterial, yeast and mammalian expression systems is the abundant expression of recombinant proteins, which are in many cases antigenically, immunologically and functionally similar to their authentic counterparts. In addition, baculoviruses are not pathogenic to vertebrates. In this report, we describe the expression and purification of ram p25 by using this expression system and analyze the biochemical properties of the purified ram p25.

Materialsand Methods Materials. c-Ha-ras p21-expressed in Escherichia coli, which was a gift from Dr. S. Hattori (National Institution of Neuroscience, Tokyo, Japan) and c-Ha-ras p21 was expressed abundantly and purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. GTP, G T P y S and G D P were purchased from Boehringer-Mannheim. [35S]GTPyS (spec. act. 1262 Ci/ml), [7-32p]GTP (spec. act. 30 C i / m l ) and [8,5'3H]GDP (spec. act. 9.2 C i / m l ) were from Du Pont-New England Nuclear. DEAE-Toyopearl 650 (S) and HCA100S were obtained from Tosoh and Koken, respectively. Other materials and chemicals were from commercial sources. Plasmid construction. To express ram p25 in Sf9 cells, we used a baculovirus transfer vector, pAcYM1, which includes baculovirus polyhedrin gene promoter [17], following the scheme outlined in Fig. 1. The ram

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cDNA constructed in pUC118 was digested with StuI and B a m H I linker was added to the StuI site. Then, the plasmid was digested with B a m HI to isolate the 0.9-kbp fragment containing the entire coding sequence of the ram protein. This fragment was inserted into the B a m H I site of pACYM1. Transfection and selection o f recombinant t'it~us. 1 p.g

of the viral DNA was mixed with 12.5-25 p.g of the plasmid r a m / p A c Y M l in 950 p.l of Hepes buffer (20 mM Hepes, 1 mM Na2HPO 4, 5 mM KC1, 140 mM NaCI, 10 mM glucose (pH 7.05)). After precipitation with 50 p.l of 2.5 M CaCI 2, the DNA segment was placed onto a monolayer culture of Sf9 cells and the culture was incubated for 1 h at room temperature. Then the supernatant of the culture was replaced by 2 ml of Grace's medium containing 10% fetal bovine serum. After incubation at 27°C for 4 days, the supernate was diluted 10- to 1000-fold and subjected to a plaque assay. The viruses that had undergone recombination could be visually screened because the recombinant viruses formed transparent plaques (due to the lack of polyhedra) and were distinct from the wild-type viruses, which formed white plaques. The recombinant viruses thus isolated were purified [18]. [35S]GTPyS-binding actiL,ity assay. Unless otherwise indicated, [35S]GTPyS-binding activity was determined with minor modifications of the rapid filtration technique [19]. Method I: Samples were incubated for various periods of time at 30°C in 100 /~I of the reaction mixture containing 20 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.0965 mM MgSO 4 and 1 p.M [35S]GTPyS (1000-2000 c p m / p m o l ) . In the reaction mixture, the free Mg 2+ concentration was calculated to be 10 p,M. The reaction was stopped by the addition of 3 ml of ice-cold stop-solution (20 mM Tris-HCl (pH 7.5), 100 mM NaCI, 25 mM MgCl2), followed by rapid filtration on a nitrocellulose filter. The filter was washed five times with the same buffer. After the filter had been dried, the radioactivity on the filter was counted in 6 ml of scintillation mixture. Method H: Samples were incubated for the indicated periods of time at 30°C in 100 p.l of the reaction mixture containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 25 mM MgCI 2, 1 mM DTT, 100 mM NaCl, 0.1% Lubrol, 250 mM (NH4)2SO 4 and 1 p.M [35S]GTPyS (1000-2000 cpm/pmol). The reaction was terminated in the same way as in Method I. [y-3ep]GTP-hydrolysis assay. The [y-32p]GTP-hydrolysis assay was performed by the essentially same method as described in Ref. 20. Purified ram p25 (3 pmol active protein estimated by GTPyS-binding) was incubated at 30°C for 30 rain in 50 p.l of buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCI 2, 10 mM EDTA, 1 mM D T T and 1 p.M [y-32p]GTP (4000-5000 c p m / p m o l ) . Then, the [y-32p]GTP-hydrolysis reaction

164 was started by adding GTP and MgCI2 to a final concentration of 0.5 mM and 5 raM, respectively. At the indicated times the reaction was terminated in the same way as in the [35S]GTPTS binding assay and the radioactivity on nitrocellulose filter was counted. [ 3H]GDP- and [ 35S]GTPyS-dissociation assay. The [3H]GDP-dissociation assay was also performed with minor modifications of the rapid filtration technique [21]. Purified ram p25 (3 pmol active protein estimated by GTPyS-binding) was incubated at 30°C for 30 rain a 25 /xl of GDP exchange buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCI;, 10 mM EDTA, 1 mM DTT and 1 g M [3H]GDP (9000-11000 c p m / p m o ! ) . Then, the [3H]GDP dissociation was initiated by adding of 25/zl of 20 mM Tris-HCl (pH 7.5) containing 1 mM DTT, 15 mM MgCI2 and 0.5 mM GTP. The rate constant for GDP dissociation was measured by displacement of [3H]GDP from its complex with rant p25. At the indicated time points the reaction was stopped in the same way as the [35S]GTPyS-binding assay and the radioactivity on nitrocellulose filter was counted. Kinetics of [35S]GTPTS dissociation were determined as for GDP dissociation, except that [35S]GTPyS (4000-5000 c p m / p m o l ) and G T P y S were used instead of [3H]GDP and unlabelec~ GDP. Other assays. SDS-PAGE was performed according to the method of Laemmli [22] and protein bands were visualized by Coomassie blue stain. Protein concentrations were determined by the method of Bradford [23] with bovine serum albumin as a standard protein.

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Expression and purification of ram p25 Sf9 ceils were transfected with recombinant virus. After 3 days. the appearance of a new 25-kDa protein band in the cells was examined by SDS-PAGE (data not shown), the cells (1.5" 10 ~ cells) were collected by centrifugation and resuspended in 10 ml of buffer A (20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). Then, the ceils were disrupted by sonication on ice for a total of 3 min with 15-s bursts in a probe-type sonicator (Branson-sonifier B12) and the suspension was subjected to centrifugation at 105000 × g for 1 h at 4°C. The supernatant was dialyzed overnight against 5 liters of buffer B (20 mM Tris-HCl (oH 7.5), 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF). The dialyzed sample was applied to a DEAEToyopearl 650 (S) column ( 1 . 6 x 10 cm) previously equilibrated with buffer B. After washing with the same buffer, the proteins were eluted at a flow rate of 2 m l / m i n with a linear gradient of NaCI from 0 to 200 mM for 30 min, using a Pharmacia FPLC system. When the [35S]GTPyS-binding activity was assayed by using Method I, the activity eluted approximately at

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Fig. 2. DEAE-Toyopearl 650 (S) and HCA-100S column t hromatography of r a m p25. (A), DEAE-Toyopearl 650 (S); (B), HCA-100S; (C), SDS-PAGE analysis of the fractions eluted from HCA-100S. Proteins eluted from the column were collected in 1.5 ml (A) and 1.0 ml (B) fractions. [35S]GTP~S-binding activit3, (e) of 5 p,l of each fraction was assayed by Method I1 as described in Materials and Methods. Fractions eluted from the HCA-100S column were analyzed by S D S - P A G E (13.5% polyacrylamide) and proteins were visualized with Coomassie blue stain; the number of each lane corresponds to the fraction number. The markers used were phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), trypsin inhibitor (20100) and a-lactalbumin (14400).

100 mM NaC1, as shown in Fig. 2A. These active fractions of DEAE-Toyopearl were collected and applied to a hydroxyapatite HCA-100S column (1 x 11 cm), previously equilibrated with buffer B containing 100 mM NaCI. After washing with the equilibration buffer, elution was performed at a flow rate of 1 m l / m i n with a linear gradient of potassium phosphate

165 from 0 to 30 mM for 60 min. When each fraction was assayed for [35S]GTPyS-binding activity, one active peak eluted at about 10 mM potassium phosphate (Fig. 2B) and the active fraction was found to contain a nearly homogeneous protein with M~ 25000 by SDSPAGE analysis (Fig. 2C). These fractions of HCA-100S containing ram p25 were collected and concentrated to more than 1 mg/ml by centrifugation using Centricon 10. The final preparation was stored at -80°C. Thus, we purified the expressed rant p25 by two steps of column chromatography, as shown in Fig. 3 and finally 504 #g of ram p25 was purified from 3.87 mg of the cytosolic fraction of Sf9 cells. On the other hand, in the membrane fraction of Sf9 cells, the ram p25 was also found by SDS-PAGE analysis, but [35S]GTPyS binding activity was not detected. This protein, which was detected by SDS-PAGE analysis, was not solubilized in either 10% cholate or 10% Triton X-100. Guanine nucleotide-bmding cttaracteristics o f ram p25

Fig. 4 shows the time-course of [35S]GTPyS-binding to ram p25. In 10 ~M Mg 2+, the [35S]GTPyS-binding activity of ram p25 increased to a steady-state in 20 min and continued this state for 30 min. The ras p21 bound [3sS]GTPyS in a time-dependent manner like ram p25. While the concentration of Mg "~+ was adjusted to 5 mM, the rate and capacity of [35S]GTPySbinding decreased greatly. On the other hand, when

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TIME (min) Fig. 4. Time-course studies of [35S]GTPyS-binding to r a m p25 and p21. About 75 ng r a m p25 (121, ©, e) and ras p21 (zx) was incubated with [35S]GTPyS according to Method I as described in Materials and Methods. The reaction was carried out at 30°C in 10 # M Mg 2- ( r n ~ ) and 5 mM Mg 2+ in the absence (,2,) and the presence (e) of 250 m M (NH4)2SO 4. At indicated times, [35S]GTPyS-binding activity to r a m p25 or r a s p21 was measured as described in Materials and Methods. ras

250 mM (NH4)2SO 4 was added to this reaction mixture containing 5 mM Mg z+, the rate of [35S]GTP~,Sbinding to ram p25 accelerated significantly. This strongly suggested that purified ram p25 binds GDP and addition of (NH4)zSO 4 to the reaction mixture causes the release of GDP from the protein as well as from a heterotrimeric G-protein, Go, probably due to the conformational change in the vicinity of guanine nucleotide-binding sites of these proteins [24]. The purified preparation of ram p25 bound [35S]GTPyS in a dose-dependent manner, as shown in Fig. 5A. When the concentration of GTPyS was increased, the [3-~S]GTPyS binding increased in a linear fashion up to 0.3 p,M and appeared to reach a plateau at 1.3 ~M GTPyS. The ras p21 bound [35S]GTPyS in a dose-dependent manner like ram p25. Scatchard plot analysis revealed that this protein bound maximally 0.8 + 0.02 tool of GTPyS nucleotide/mol of protein with a K d value of 340 + 4.91 nM (means _+ S.E.) and that ras p21 bound maximally 0.7+_0.03 mol of G T P y S / m o l of protein with a K d value of 280 +_2.25 nM (means _+ S.E.) under the condition as shown in Fig. 5B. The dissociation f r o m ram p25

rate o f [L~H]GDP and [ 3 5 S ] G T P y S

In order to ascertain the kinetics of dissociation of [3H]GDP from ram p25, the protein was preincubated with [3H]GDP at 30°C for 30 min. Then, the second incubation was performed by adjusting the concentration of Mg 2- and GTP to 5 and 0.5 raM, respectively,

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Fig. 5. Concentration dependence of [35S]GTPyS-binding to ram p25 and ras p21. About 300 ng r a m p25 (A) and ras p21 (B) was incubated at 30°C with various concentrations of [35S]GTPyS according to Method II as described in Materials and Methods. After 15 rain incubation, [35S]GTPyS-bindingactivity to r a m p25 and ras p21 was measured as described in Materials and Methods. The insets show the Scatchard plots. The results are the representative of three independent experiments.

and the p r o t e i n - b o u n d radioactivity was d e t e r m i n e d at various times. T h e release of [ 3 H ] G D P at 30°C occurred with 0.015 + 0.0010 m i n - ~ ( m e a n s + S.E.) with a half-time of 46 m i n (Fig. 6). T h e a d d i t i o n of 250 m M ( N H 4 ) 2 S O 4 greatly increased this dissociation rate to 0.41 + 0 . 0 0 1 5 m i n - t ( m e a n s - + S.E.). T h e dissociation rate of r a s p21 was observed to be 0.008 -+ 0.0058 a n d

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0.23 + 0.058 m i n - ~ ( m e a n s + S.E.) in the presence of 5 m M Mg 2+ or 250 m M (NH4)2SO4, respectively. T h e release of [35S]GTP3,S was observed to be 0.004 + 0.0010 m i n -1 ( m e a n s + S.E.) u n d e r the same e x p e r i m e n t a l c o n d i t i o n s as e m p l o y e d for [ 3 H ] G D P release (data not shown), r a m p25 was p r e i n c u b a t e d with [35S]GTP3'S for 30 m i n a n d t h e n the c o n c e n t r a t i o n of Mg ~+ a n d G T P 3 ' S was adjusted to 5 a n d 0.5 mM, respectively, a n d the p r o t e i n - b o u n d radioactivity was d e t e r m i n e d at various times. It was shown that r a m p25 had a higher affinity for G T P t h a n G D P . [3'-32p]GTP-hydrolysis

rate

T h e intrinsic G T P a s e activity of purified r a m p25 was m e a s u r e d by the released [32p]p i from the complex of [3'-32p]GTP a n d r a m p25 as described in Materials a n d M e t h o d s ' . As shown in Fig. 7, the hydrolysis rate for r a m p25 was calculated to be 0.010 _+ 0.0012 m i n ( m e a n s _+ S.E.). T h e rate of r a s p21 was observed to be 0.020 _+ 0.0010 min ~ ( m e a n s -+ S.E.) u n d e r the same e x p e r i m e n t a l conditions.

60

TIME (min) Fig. 6. Measurements of the dissociation rate of [3H]GDP from

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Fig. 7. Measurements of the [7-32p]GTP-hydrolysisrate of r a m p25 and ras p21. Active 3 pmol rant p25 (o) or ras p21 (z~) was incubated at 30°C with 1 p.M [T-32p]GTP as described in Materials and Methods. After 30 rain incubation, Mg2+ (5 mM of final concentration) was added and the [y-32p]GTP-hydrolysis reaction was started by adding excess GTP (final concentration 500/xM). At indicated times, the reaction was stopped and [7-32p]GTP bound to the protein was measured as described in Materials and Methods. The results are the representative of three independent experiments.

The

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p25 and ras p21. Active 3 pmol r a m p25 (O, e)or ras p21 (zx. • ) was incubated at 30°C with 1 lzM [3H]GDP, as described in Materials and Methods. After 30 rain incubation, the dissociation reaction of [3H]GDP was started by adding excess GTP (final concentration 500 lzM) in the presence (*, • ) and the absence (o, zx) of 250 mM (NH4)2SO4. At indicated times, the reaction was stopped and the [3H]GDP bound to the protein was measured as described in Materials and Methods. The results are the representative of three independent experiments.

Discussion With the aid of the baculovirus expression system, which allowed the p r o d u c t i o n at high level of soluble r e c o m b i n a n t proteins, we could express r a m p25 in insect cells in large a m o u n t s . T h e a p p e a r a n c e of this p r o t e i n b a n d in both the cytosolic a n d the m e m b r a n e fraction was assessed by S D S - P A G E (data not shown).

167 The [35S]GTPyS-binding activity was not detected in the membrane fraction, and also the protein, which was detected by SDS-PAGE analysis, could not be solubilized by detergents tested as described in Resuits. These results suggested that the r a m p25 expressed in the membrane fraction might be a denatured form which could not be released from membranes. On the other hand, r a m p25 expressed in the cytosolic fraction had a high [35S]GTPyS-binding activity and was purified by column chromatography. The availability of high amounts of active purified protein allowed us to investigate the detailed biochemical characterization of r a m p25. We demonstrated here that the purified r a m p25 binds guanine nucleotide and has a low GTP-hydrolysis activity. This protein was stable in the presence of 10 mM Mg 2÷ which prevents the dissociation of bound GDP. By decreasing the Mg 2÷ concentration with EDTA to 5 ~M, the dissociation rate increased and the exchange reaction accelerated. These results indicate that the Mg 2+ probably regulates the conformation of r a m p25 as observed in ras p21: in the presence of millimolar Mg 2+, the r a m p25-GDP complex exchanges the bound nucleotide with exogenous nucleotides very slowly. When Mg 2+ is depleted, the complex is reformed and undergoes free exchange with exogenous nucleotides [25]. The addition of ammonium sulfate (final 250 mM) also enhanced the exchange reaction by facilitating the dissociation of bound GDP from r a m p25 as well as ras p21. Thus, ammonium sulfate appears to cause a conformational change of r a m p25 in the guanine nucleotide-binding site on the vicinity of the protein [26]. In time-course and concentration-dependent studies, the purified r a m p25 bound [35S]GTPyS in a similar manner to that of ras p21. However, some biochemical properties of the two proteins were different. The [3H]GDP-dissociation rate of r a m p25 was approx. 2-times higher than that of ras p21 ( r a m p25, 0.015 min-l; ras p21, 0.008 min-t). Also, the rate of [y-32p]GTP-hydrolysis was lower in r a m p25 than ras p21 ( r a m p25, 0.010 m i n - l ; ras p21, 0.020 min-l). These observations indicate that the rate-limiting step is the GTP-hydrolysis reaction in the guanine nucleotide turnover in r a m p25, while the limiting step in ras p21 is the GDP-dissociation reaction [21]. Since the GDP-dissociation and GTP-hydrolysis rates of low M r GTP-binding proteins are very low, it has been suggested that some regulatory components modulating guanine nucleotide exchange or GTP-hydrolysis reaction were present. Indeed, four types of component have been found for several low M r GTPbinding proteins, which stimulate or inhibit intrinsic GTP-hydrolysis or GDP-dissociation [1]. A GTPase activating protein (GAP), which is capable of increasing the GTP-hydrolysis activity of r a s p21 more than

100-fold, has been identified in X e n o p u s oocyte and a wide range of mammalian cells [27,28]. A GTPase inhibiting protein for ras p21 has also been detected in brain cytosolic fraction [29]. Furthermore, other types of regulatory factor which affect GDP-dissociation have been found; GDP-dissociation stimulator (GDS), stimulating GDP-dissociation and subsequent GTP-binding [30,31] and GDP-dissociation inhibitor (GDI), inhibiting GDP-dissociation [32,33]. In the present study, we have measured the intrinsic GTPase and GDP-dissociation activities of the pure preparation of r a m p25 expressed in baculovirus system, r a m p25 offers an advantage to search components which functionally interact with it and to examine their effects on its catalytic properties. Actually, the GDI activity for r a m p25 was recently found to be present in the cytosol of rat spleen [34]. From the sequence comparison, the r a m p25 has a very similar putative effector domain in S E C 4 protein (ram p25; FITTVGIDFR 47, S E C 4 protein; FIT-I'IGIDFK57), and shares 40% amino-acid identity and 60% homology with S E C 4 protein [13,35]. This leads us to speculate that r a m p25 might play an important role in some step(s) of vesicular transport in platelets as observed to S E C 4 protein in S a c c h a r o m y c e s c e r e cisiae [13].

Acknowledgements We thank Dr. S. Hattori (National Institute of Neuroscience, Tokyo) for supplying c - H a - r a s p21-expressing E s c h e r i c h i a coli. This work was in part supported by the grants from the Ministry of education, Science and Culture of Japan, and from Yamanouchi Foundation for Research on Metabolic disease.

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