Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 49:147–155 (2002) Purification and Characterization of Nucleoside Diphosphate Kinase From the Brain of Bombyx mori Tomohide Uno,* Mayumi Ueno, Michiko Kikuchi, and Yasuo Aizono Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity. Arch. Insect Biochem. Physiol. 50:147–155, 2002. © 2002 Wiley-Liss, Inc. KEYWORDS: NDP kinase; brain; Bombyx mori INTRODUCTION Nucleoside diphosphate kinase (NDP kinase, EC 188.8.131.52) catalyzes the transfer of a phosphoryl group from nucleoside triphosphate to nucleoside diphosphate [N1TP+N2DP «N1DP+N2TP] (Parks and Agarwal, 1973). NDP kinase is not substrate specific and can use purine and pyrimidine, riboor deoxy ribonucleotides as substrate. Previously, the primary role of NDP kinase in the cell was considered as the maintenance of a pool of nucleoside triphosphates required for biosynthesis, but lately several NDP kinases encoded by the genes of the nm23 family have been shown to play important roles in tumor metastasis, morphogenesis, cell proliferation, differentiation, and transcriptional regulation (Lacombe et al., 1992; Rosengard et al., 1989: Biggs et al., 1990; Keim et al., 1992; Lakso et al., 1993; Okabe-Kado et al., 1988; Postel et al., 1993). For example, nm23-H1 has been reported to be down regulated in some metastic cell lines (Wallet et al., 1990). The gene, nm23-H2, was identified as a transcriptional factor for c-myc, a well-known regulator of cell proliferation and differentiation (Stahl et al., 1991; Postel et al., 1993). In insects; the product of the abnormal wing disc (awd) developmental gene, which is responsible for normal wing development in Drosophila melanogaster, exhibits NDP kinase activity (Dearolf et al., 1988). Little is known about the biochemical and functional features of NDP kinase of insects except Drosophila melanogaster (Inoue et al., 1996). In this study, NDP kinase was purified to homogeneity from the brain of Bombyx mori. The purified protein was demonstrated to be NDP kinase, as judged from the partial amino acid sequence. The purified protein showed NDP kinase activity Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe Hyogo, Japan Abbreviations used: NDP kinase = nucleoside diphosphate kinase; BSA = bovine serum albumin; PTTH = prothoracicotropic hormone; MBP = myelin basic protein; GTPgS = guanosine 5,-O-(3-thiotri) phosphate. Grant sponsor: Ministry of Education and JSPS Research for the Future Program. Grant number: 14760033. *Correspondence to: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe Hyogo 657-8501, Japan. E-mail: firstname.lastname@example.org Received 19 April 2001; Accepted 16 March 2002 © 2002 Wiley-Liss, Inc. DOI: 10.1002/arch.10037 Published online in Wiley InterScience (www.interscience.wiley.com) 148 Uno et al. and phosphorylated myelin basic protein (MBP) in vitro. Dephosphorylation of the purified NDP kinase increased NDP kinase activity. MATERIALS AND METHODS Materials Pupae of the silkworm, Bombyx mori, was purchased from Nikko Shoji. DEAE-Cellulofine A-500m and hydroxyapatite were donated by Seikagaku Kogyo Co., Ltd. Lysyl endopeptidase was purchased from Wako Pure Chemicals Industries. Mono-Q HR5/5 and Superdex 75 PC 3.2/30 were purchased from Pharmacia LKB. [35S] GTPgS (1,000– 1,500 Ci/m mol) and [g-32P] ATP (3,000 Ci/m mol) were from ICN Biochemicals. Other chemicals used were of analytical grade. Determination of Protein Proteins were determined using BSA (Fraction V, Sigma) as the standard by the method of Lowry et al. (1951). SDS-PAGE SDS-polyacrylamide gel analysis was performed according to the method of Laemmli (1970), using a 4.5% stacking gel and a 15% separating gel, at a constant current of 16 mA. The proteins in a gel were stained by the silver-staining method (Morissey 1981). GTPgS Binding Assay The incubation mixture included 50 mM TrisHCl (pH 8.0), 40 mM [35S] GTPgS, and 10 mM MgCl2 in a volume of 100 ml. The reaction was allowed to proceed for 30 min at 25°C and stopped by adding 1 ml of washing buffer (20 mM TrisHCl, pH 8.0, 25 mM MgCl2, and 0.1 M NaCl). The reaction mixture was filtrated through a nitrocellulose membrane filter. The membrane was washed twice with 1 ml of washing buffer and dried. The filter-bound radioactivity was counted by a liquid scintillation counter (Aloka, LSC-5100). Protein Sequence Analysis For determination of the partial amino acid sequences, the purified enzyme (30 mg, 1.8 nmol) was incubated with 0.3 mg of lysyl endopeptidase at 30°C for 16 h in 100 ml of 50 mM Tris-HCl (pH 9.0) containing 4 M urea. The mixture was subjected to reverse phase high performance liquid chromatography using a mBondasphere C18 column equilibrated with 0.1% (w/v) trifluoroacetic acid. Peptide fragments were separated with a linear-gradient of acetonitrile, from 0 to 60% (v/v). The amino acid sequences were determined by automated Edman degradation using a Shimadu PPSQ-10 protein sequencer. NDP Kinase Assay Nucleotide diphosphate kinase assay was determined as follows. The standard assay (40 ml) contained 5 mM [g-32P] ATP (10 mCi), 1.5 mM GDP, 5 mM MgCl2 and 50 mM MES-NaOH (pH 6.0). Reaction was started by adding 10 ng of purified protein, carried out at 30°C for 2.5 min, and stopped by adding 40 ml of 50 mM GTP. Ten-microlitter aliquots of the samples were spotted on polyethyleneimine-cellulose sheets and then developed in 0.75 M potassium phosphate buffer (pH 3.4) for 90 min at room temperature. Reaction products were located under an ultraviolet lamp. The radioactivities were counted using an imaging analysis, BAS 1000 Man (Fuji Film Co., Tokyo, Japan). One unit was defined as the amount of enzyme that catalyze the production of 1 mM GTP min–1. Protein Kinase Assay The mixture with a final volume of 20 ml contains 20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10 mM [g-32P] ATP (0.1 mCi), and 1 mg of the protein. The phosphorylation was started by adding 5 ml (10 ng) of NDP kinase to 20 ml of the reaction mixture. After 60 min incubation at 25°C, the reaction was terminated by adding 4 ml of SDS-sample buffer. The samples were subjected to SDS-PAGE, and dried. 32P-labelled Archives of Insect Biochemistry and Physiology Purification of NDP Kinase bands were visualized by exposing the gel to Kodak X-Omat films. Gel Filltration The purified protein (7.8 mg, 40 ml) was applied to a Superdex 75 PC 3.2/30 column (Smart system, Pharmacia) equilibrated with 62.5 mM TrisHCl (pH 6.7) and 0.1 M NaCl at a flow rate of 40 ml/min. The standard proteins are as follows; myoglobin (17.6 kDa), chymotrypsinogen A (25.6 kDa), ovalbumin (45 kDa), and BSA (67 kDa). Activity of Dephosphorylated and Autophosphorylated NDP Kinase Purified enzyme (10 ng) was dephosphorylated at 30°C for 10 min in 10 ml of reaction mixture A [20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, and 6 milli units of bacterial alkaline phosphatase]. The NDP kinase reaction was started by adding 25 ml of reaction mixture B [50 mM MES-NaOH (pH 6.0), 10 mM Na3VO4, 5 mM MgCl2, 5 mM [g-32P] ATP and 1.5 mM GDP]. Reactions were incubated at 30°C for the indicated period of time. The reaction was stopped with the addition of 50 mM GTP. The reaction products were analyzed as described previously. The NDP kinase reaction mixture of autophosphorylated NDP kinase was made by adding 1 mM ATP in place of alkaline phosphatase in reaction mixture A. As control, NDP kinase was incubated in reaction mixture A omitting bacterial alkaline phosphatase and reaction mixture B. RESULTS AND DISCUSSION Purification of NDP Kinase The method to assay NDP kinase activity using thin layer chromatography is time-consuming and difficult. The GTPgS [Guanosine 5¢-O-(3-thiotri) phosphate], which is GTP analog, filter binding assay was the easy and conventional method to detect NDP kinase indirectly (Inoue et al., 1996). NDP kinase binds [35S] GTPgS, transfers the [35S] -thiol phosphate to NDP kinase, and [35S] -thiol July 2002 149 phosphate-bound NDP kinase is trapped on nitrocellulose membrane (Inoue et al., 1996). So, we used [35S] GTPgS filter binding assay to purify NDP kinase from the brain of Bombyx mori and identified the purified protein as NDP kinase by protein sequencing. All procedures were carried out at 4°C. Brains of pupae (11.84 g) were homogenized in buffer A [20 mM Tris-HCl (pH 8.0), 0.25 M sucrose, 1 mM EDTA, 1 mM Diisopropyl flurophosphate, 1 mM DTT, and 2 mg/ml pepstatin A]. The homogenate was centrifuged at 20,000g for 30 min. To the supernatant, solid ammonium sulfate was added to 70% saturation, followed by centrifugation. The precipitate was dissolved in 12 ml of buffer A and dialyzed against buffer A for three days. After centrifugation, the supernatant was applied to a DEAE-Cellulofine A-500 (f1.5 ´ 24 cm) equilibrated with buffer A. After washing with buffer A, proteins were eluted with a step-wise increase in NaCl (Fig. 1). GTPgS binding activity was mainly recovered in 0.03 M NaCl and 0.2 M NaCl eluates. The 0.03 M NaCl eluate was dialyzed against buffer B [10 mM potassium phosphate (pH 7.2), 1 mM DTT, and 0.1 mM EDTA]. After centrifugation, the supernatant was applied to a hydroxyapatite column (f1.0 ´ 10 cm) equilibrated with buffer B. After washing with buffer B, proteins were eluted with a step-wise increase in potassium phosphate. GTPgS binding activity was recovered in 0.04 M potassium phosphate. The eluate was dialyzed against buffer C [20 mM sodium acetate (pH 5.5)]. After centrifugation, the supernatant was applied to a Mono-S HR5/5 column equilibrated with buffer C. After washing with buffer C, the adsorbed proteins were eluted with a linear gradient of 0-0.5 M NaCl in buffer C (Fig. 2A). GTPgS binding activity was detected between 0.04 to 0.07 M NaCl on the linear gradient. The active fractions were pooled and dialyzed against buffer D [20 mM Tris-HCl (pH 8.3)]. After centrifugation, the supernatant was applied to a Mono-Q HR5/5 column equilibrated with buffer D. After washing with buffer D, the adsorbed proteins were eluted with a linear gradient of 0–0.5 M NaCl in buffer D (Fig. 2B). GTPgS binding activity was detected between 0.05 to 0.09 150 Uno et al. Fig. 1. DEAE-Cellulofine column chromatography. The crude enzyme preparation was applied to a column of DEAE-Cellulofine (f1.5 ´ 24 cm), washed, and then eluted stepwise with NaCl. The flow rate was 1.5 ml/min. Solid line, absorbance profile at 280 nm. Dotted line, GTPgS binding activity. M NaCl on the linear gradient . A typical purification procedure is summarized in Table 1. Finally, 55 mg of GTPgS binding protein with a binding constant of 0.36 nmol was purified from 11.8 g of the brain of Bombyx mori. The sequential steps gave an approximately 136-fold augmentation in specific content with a yield of 3.6%. As shown in Figure 3A, the purified protein migrated as a single band on SDS-PAGE. The estimated molecular mass on SDS-PAGE was 18 kDa. tain whether the purified protein was NDP kinase or not, the NDP kinase activity was measured. The purified protein showed NDP kinase activity. Optimal activity was observed at pH 6.0. Protein Sequence of NDP Kinase The 18-kDa protein was digested with lysyl endopeptidase, and the amino acid sequences of resulting peptides were determined. The amino acid sequences were compared with those present in the GenBank nucleotide database (by using the BlastN algorithm). The B. mori protein showed high similarity to the deduced amino acid sequence of the Drosophila NDP kinase (Fig. 4). Next, to ascer- Enzymatic Properties of NDP Kinase NDP kinase of Drosophila was found to function as a protein kinase (Inoue et al., 1996). So, various exogenous protein substrates were tested as possible substrates for phosphorylation in vitro. When myelin basic protein (MBP) was incubated with purified NDP kinase and [g-32P] ATP, incorporation of radioactivity into the protein was detected (Fig. 3B, lane 3). NDP kinase was also faintly autophosphorylated (Fig. 3B, lane 1). BSA, BRab (Uno et al, 1998) and histone were not phosphorylated (data not shown). MBP is a protein constitutent of myelin, nerve sheath, and used as a marker protein of oligodendroglial cells (Shepard, 1994). NDP kinase in the brain of Bombyx mori Archives of Insect Biochemistry and Physiology Purification of NDP Kinase Fig. 2. Mono-S column chromatography (A) and MonoQ column chromatography (B). The fractions were applied to a column of Mono-S HR5/5 (f1.6 ´ 50 mm) and MonoQ HR5/5 (f1.6 ´ 50 mm), respectively, washed, and then eluted with a 3-ml gradient of 0 to 0.5 M NaCl and with July 2002 151 a 2-ml gradient of 0 to 0.3 M NaCl, respectively. Solid line, dotted line, and dashed line, absorbance profile at 280 nm, GTPgS binding activity, and elution gradient of NaCl, respectively. 152 Uno et al. TABLE 1. Summary of NDP Kinase Purification From the Brain of Bombyx mori Step Cruse extract 70% (NH4)2SO4 DEAE-cellulofine Hydroxyapatite Mono-S Mono-Q Total protein (mg) 203 194 23.1 9.05 0.098 0.055 Total GTP g S binding (pmol) 9,720 9,030 8,470 4,640 374 357 Specific binding (pmol/mg) 47.9 46.5 367 512 3,820 6,490 Yield (%) 100 92.8 87.1 48.3 3.85 3.67 Purification (fold) 1.00 0.971 7.66 10.6 79.7 136 may regulate the connection between the neuron and the glial cell by phosphorylating MBP-like protein in insect brain. All known NDP kinase are oligomeric and made of small polypeptides of about 150 residues (molecular mass of about 17 kDa) with a high degree of sequence similarity (Hemmerich and Pecht, 1992). On a gel filtration column, the purified Fig. 3. SDS-PAGE analysis of NDP kinase purified from the brain of Bombyx mori (A) and phosphorylated proteins (B). A: Lane 1: molecular mass marker. Lane 2: purified NDP kinase (1 mg). B: MBP (1 mg) was incubated (60 min, 25°C) with purified NDP kinase (10 ng) and 10 mM [g32 P] ATP (0.1 mCi) in a reaction buffer [20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM EDTA and 1 mM DTT] (Lane 3). NDP kinase was autophosphorylated with [g32 P] ATP in a reaction buffer (Lane 1). Then, the reaction was terminated by adding SDS-sample buffer. The samples were subjected to SDS-PAGE. As control, MBP was reacted with [g-32P] ATP in a reaction buffer (Lane 2). Archives of Insect Biochemistry and Physiology Purification of NDP Kinase 153 Fig. 4. Comparison of the deduced amino acid sequences of Bombyx mori NDP kinase peptides with the sequences of Drosophila melanogaster NDP kinase. Identical amino acids are indicated by asterisks. protein exhibited a molecular mass of 67 kDa. From this result, it appears that NDP kinase in the brain of Bombyx mori associate to form a tetrameric structure. To examine the interaction among subunits, Hill plots of NDP kinase were constructed. The values for Km and n were calculated to be 31.6 mM ± 0.1 and 1.14 ± 0.03, respectively. The n value so close to 1.0 indicates little or no cooperativity on interaction among subunits of NDP kinase from the brain of Bombyx mori. The purified NDP kinase bound GDP with a higher affinity, compared to the other NDP kinase with reported Km values, 125 mM (Ulloa et al., 1995). autophosphorylated NDP kinase is suggested not to be a phosphorylatted ntermediate during NDP kinase GDP-phosphorylating reaction. NDP kinase activities of autophosphorylated- and dephosphorylated forms, respectively, were measured (Fig. 5). The basal and the autophosphorylated NDP kinsae showed no significant differences in activity (Fig. 5; solid squares and triangles). NDP kinase activity was increased by dephosphorylation (Fig. 5; open squares). These results suggest that autophosphorylation of NDP kinase inhibits the NDP kinase GDP-phosphorylating reaction and the autophosphorylated form is not an intermediate during the NDP kinase GDP-phosphorylating reaction. Both NDP kinase GDP-phosphorylating activity and autophosphorylation activity were affected by magnesium ions. The maximal activities of autophosphorylation and GDP-kinase activity were observed at 0.1 mM (n = 3) and 7 mM (n = 3) MgCl2, respectively. These results support the proposal that autophosphorylation and GDP-kinase activity are different reactions. In the brain of insects, some protein phosphatase may regulate the function of the phosphorylated NDP kinase by dephosphorylation. What role does NDP kinase have in insect brain? The enzyme has been shown to regulate various cellular functions, including tumor metastasis, morphogenesis, cell proliferation, differentia- Effect of Phosphorylation on NDP Kinase Activity NDP kinase catalyzes the transfer of the terminal phosphate from nucleotide 5¢-triphosphate to nucleotide 5¢-diphosphate (Parks and Agarwal, 1973). The mechanism of the NDP kinase reaction involves the formation of a high-energy phosphoprotein of a histidine residue (Morera et al., 1995). In the absence of NDP as acceptor, NDP kinase undergoes autophosphorylation utilizing adenosine triphosphate as phosphate donor (Hemmerrich and Pecht, 1992). Using point mutational analysis, it was shown that mutants replacing autophosphorylated amino acid residues retained NDP kinase activity (Almaula et al., 1995). So, July 2002 154 Uno et al. and GTP binding protein is responsible for effective GTP introduction into the GTP binding protein. Possibly NDP kinase in the brain is related to PTTH secretion via GTP binding protein. Nothing is known about the role of NDP kinase in the brain of Bombyx mori. Further studies are progressing to determine the functional role of NDP kinase in the brain relating to the PTTH release. ACKNOWLEDGMENT This work was supported by a grant-in-aid for Scientific Research (No. 14760033) from the Ministry of Education and JSPS Research for the Future Program. Fig. 5. NDP kinase activity of dephosphorylated- and autophosphorylated- protein. Purified enzyme (10 ng) was dephosphorylated at 30°C for 10 min in 10 ml of reaction mixture A [20 mM Tris-HCl (pH 8.0), 5 mM MgCl2 and 6 milli units of bacterial alkaline phosphatase] (open square). The NDP kinase reaction was started by adding 25 ml of reaction mixture B [50 mM MES-NaOH (pH 6.0), 10 mM Na3VO4, 5 mM MgCl2, 5 mM [g-32P] ATP and 1.5 mM GDP]. Reactions were incubated at 30°C for the indicated period of time. The reaction was stopped with the addition 50 mM GTP. The NDP kinase reaction mixture of autophosphorylated NDP kinase was made by adding 1 mM ATP in place of alkaline phosphatase in reaction mixture A (solid square). As control, NDP kinase was incubated in reaction mixture A omitting bacterial alkaline phosphatase and reaction mixture B (triangle). Y axis shows the amount of GTP produced per 10 ng NDP kinase. tion, and transcriptional regulation in mammals (Lacombe et al., 1992; Rosengard et al., 1989; Biggs et al., 1990; Keim et al., 1992; Lakso et al., 1993; Okabe-Kado et al., 1988; Postel et al., 1993). PTTH, a neuropeptide hormone producing morphological changes and ecdysis in insects, is secreted from the brain into hemolymph (Nagasawa, 1993). GTP binding proteins contribute to the release of PTTH (Shirai et al., 1998). NDP kinase has been proposed to interact with GTP binding proteins (Kimura et al., 1990). 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