Tropomyosin (TM) is one of the actin-regulatory proteins and stabilizes the filamentous structure of F-actin. Although it has been suggested that multiple TM isoforms play important roles in nonmuscle cells and several TM-binding proteins have been identified and characterized, the functional properties of nonmuscle TM isoforms are not well understood. In order to investigate the roles of TM in nonmuscle cells, we searched for novel nonmuscle TM-binding proteins from the chicken liver total acetone powder extract by TM-affinity column chromatography. As a consequence, a protein of 57 kDa was mainly eluted from the column and its partial amino acid sequence of the protease-digested fragment was determined. Homology search using SWISS-PLOT showed that the 57 kDa protein was nearly identical to chicken glutamate dehydrogenase (GDH: EC 126.96.36.199). Purified GDH had the ability to bind to the TM-affinity column, indicating that GDH associated directly with liver TM. The binding of GDH to TM was abolished in the presence of 0.1 mM ATP, that is known to decrease the rate of the GDH enzymatic activity at pH 7.5. Thus, we have demonstrated the interaction between nonmuscle TM and GDH for the first time.
Tropomyosin (TM), an α-helical coiled-coil protein, is one of the constituents of thin filaments in myofibrillar cells and of microfilaments in nonmuscle cells (Pittenger et al., 1994; Lin et al., 1997). The dimeric form of TM polymerizes in a head-to-tail manner and associates with F-actin in the two grooves of filaments to stabilize the filamentous structure. Especially in higher vertebrates, a number of TM isoforms is generated from four genes by using their alternative promoters and alternative exons. They are generally categorized into two types of high molecular weight (high Mr) and low molecular weight (low Mr) isoforms. It is well recognized that both high and low Mr TM isoforms are distributed in nonmuscular tissues as well as in embryonic muscles. As is the case of actin, isoform transition occurs from mainly low Mr isoforms to the muscle-specific high Mr ones during muscle maturation (Hosoya et al., 1989; Lin et al., 1997).
Numerous biochemical, immunocytochemical, and genetic studies have been devoted to elucidate the functional significance of nonmuscle TM isoforms and documented their involvement in cell shape stabilization, cytokinesis during cell division, intracellular granule movement, and organelle distribution (Pittenger et al., 1994; Lin et al., 1997). Thus, it is strongly suggested that TM isoforms play significant functions in nonmuscle cells. Nevertheless, those roles can not be ascribed exclusively to nonmuscle TM isoforms. TM-binding proteins are certainly involved in the regulation of these physiological events through inducing conformational changes of TM and utilizing it as a scaffold.
It is well known that TM regulates an actomyosin interaction in myofibrillar cells in association with the troponin complex in a Ca2+-dependent fashion (Ebashi et al., 1968). In nonmuscle cells, the troponin complex is absent, but several TM-binding proteins were identified. Caldesmon and calponin, that were originally isolated from smooth muscle, bind to both TM and F-actin in a Ca2+-calmodulin dependent manner (Sobue et al., 1981; Takahashi et al., 1986). Later, low Mr caldesmon and acidic calponin were detected from nonmuscular sources and characterized (Matsumura and Yamashiro, 1993; Appligate et al., 1994; Trabelsi-Terzidis et al., 1995). It is noteworthy that nonmuscle caldesmon increases the ability of low Mr TM isoforms to associate with F-actin (Yamashiro-Matsumura and Matsumura, 1988), and that it also enhances the affinity of rat nonmuscle high Mr TM-1 to F-actin, but not that of another nonmuscle high Mr, TM-2 (Pittenger et al., 1995). Although acidic calponin exhibits extensive homology in the primary structure to the smooth muscle basic calponin except for the C-terminal domain (Appligate et al., 1994), it has not been demonstrated yet whether it can bind to TM. Tropomodulin, that was firstly identified in human erythrocytes as a TM-binding protein and also found in the striated muscle, associates with the pointed-end of microfilaments (Fowler, 1996). The pointed-end capping activity of tropomodulin is greater in the presence of TM than in its absence. Other proteins that interact with nonmuscle TM, including human serum albumin (Hitchcock-DeGregori et al., 1985), myelin basic protein (Dobrowolski et al., 1986), plasma gelsolin (Koepf and Burtnick, 1992), S100 proteins (Takenaga et al., 1994; Gimona et al., 1997), were reported. Despite the existence of several proteins that can associate with TM, the physiological roles of these proteins are not fully understood.
Liver is the largest nonmuscular organ and plays central roles in diverse metabolic pathways, such as storage of glycogen, detoxification, bile acid formation and lipid turnover. Several studies have given evidences that the actin cytoskeleton is involved in the contraction of bile canaliculi in parenchymal cells and of fenestrae in nonparenchymal sinusoidal endothelial cells (Tsukada et al., 1995; Steffan et al., 1987; Gatmaitan et al., 1996). However, detailed physiological properties of microfilaments and their associated proteins in the liver parenchymal and nonparenchymal cells such as Küpffer cells and fat-storing cells are scarcely investigated and elucidated.
In this study, to obtain further information on the physiological properties of nonmuscle TM isoforms, we searched for novel nonmuscle TM-binding proteins from the chicken liver total acetone powder extract by TM-affinity column chromatography. A protein of 57 kDa, which was eluted from the TM-affinity column, was identified as an enzyme, glutamate dehydrogenase (GDH: EC 188.8.131.52). Purified GDH was also retained on the TM-affinity column in the absence of ATP, but not in its presence, indicating that GDH directly associated with chicken liver TM in an ATP-dependent manner.
MATERIALS AND METHODS
Preparation of proteins
All procedures were performed in a cold room unless otherwise mentioned. Chicken liver TM was prepared according to Tobita et al. (1996) with some modifications. Briefly, proteins were extracted from the chicken liver acetone powder with 20 volumes (v/w) of the extraction buffer containing 1 M KCl, 0.1 M KH2PO4 and 0.1% (v/v) 2-mercaptoethanol, pH 7.0 for overnight. After boiling the supernatant, isoelectric precipitation (pH 4.5) and ammonium sulfate fractionation in 40–70% saturation were carried out. The resultant precipitate was dissolved in 1 ml of 7 M urea and 0.1% 2-mercaptoethanol and dialyzed against the same solution for overnight. The dialysate was subjected to agarose-IEF according to Hirabayashi (1981). After IEF, the bands corresponding to TM were excised from the agarose gels and dialyzed against the extraction buffer for overnight. Finally, the dialysate was ultracentrifuged at 195,000 × g for 30 min using a Beckman TLA 100.3 rotor. The resulting supernatant was recovered and stored at −80°C until use.
Chicken liver glutamate dehydrogenase (GDH) was prepared as follows. The chicken liver was dissected, washed with cold PBS to remove blood and minced in 4 volumes (v/w) of the extraction buffer consisting of 1% (v/v) Triton X-100, 0.05% (w/v) cetyltrimethylammonium bromide (CTAB), 10 mM K-PO4, 1 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT, pH 7.5 using a Sorvall Mixer on ice for about 1 min. The mixture was extracted for 1 hr with stirring and centrifuged at 37,000 × g for 20 min using a Beckman JA-20 rotor. The supernatant was pooled and the resulting pellet was re-extracted in the same way as described above. These two extracts were combined and ammonium sulfate fractionation was performed in 0-30 and 30–70% saturation. The precipitate from ammonium sulfate fractionation in 30–70% saturation was dissolved in buffer I containing 10 mM K-PO4, 1 mM EDTA, 0.1 mM PMSF and 0.1 mM DTT, pH 7.5 and dialyzed against the same buffer for overnight. The dialysate was applied onto a DEAE-TOYOPEARL S650 column (TOSOH: 2.2×20 cm) equilibrated with buffer I using a FPLC System (amersham pharmacia biotech). After washing with the equilibration buffer to remove unabsorbed proteins, bound proteins were eluted with a linear concentration gradient of KCl from 0 to 0.5 M. GDH-active fractions were collected and ammonium sulfate fractionation in 0–70% saturation was done. The pellet was dissolved in 1 M ammonium sulfate in buffer I, pH 7.5 and applied onto a Butyl-TOYOPEARL S650 column (TOSOH: 2.2×20 cm) equilibrated with the same buffer. After washing with the starting buffer, adsorbed proteins were eluted with a decreasing linear concentration gradient of ammonium sulfate from 1 to 0 M. GDH was eluted at the concentration between 0.25 and 0 M ammonium sulfate. These fractions were subjected to ammonium sulfate fractionation. The centrifugal pellet was suspended, dialyzed against buffer I, and applied to a Hydroxylapatite column (BIO-RAD: 1.6×15 cm) equilibrated with buffer I. After washing the column, GDH-containing fractions were eluted with a linear concentration gradient of K-PO4 from 10 to 500 mM. GDH-containing fractions were desalted, dialyzed, and loaded onto a HiTrap Blue affinity column (amersham pharmacia biotech) equilibrated with buffer I. After washing, bound proteins were eluted at 3 steps with different concentrations of KCl, 0–0.9 M, 0.9–2 M and 3 M in buffer I. GDH was eluted at a concentration of KCl more than 0.9 M and these fractions were concentrated and desalted with 0.15 M KCl in buffer I, pH 7.5 using Centriprep-10 (Amicon). The GDH-enriched fraction was applied onto a HiLoad Superdex 200 pg column (amersham pharmacia biotech: 1.6×60 cm) pre-equilibrated with 0.15 M KCl in buffer I, pH 7.5 and eluted with the same buffer.
Actin was prepared from a rabbit skeletal muscle according to Spudich and Watt (1971).
Determination of protein concentration
Concentrations of chicken liver TM and GDH were determined using A280=0.24 for 1 mg/ml of platelet TM (Côte and Smillie, 1981) and A280=0.95 for 1 mg/ml of chicken liver GDH (Frieden, 1962), respectively. Those of other proteins including crude fractions of TM or GDH were calculated using A280=0.63 for 1 mg/ml of BSA as a standard.
Electrophoresis and immunoblotting
SDS-PAGE was performed by the method of Laemmli (1970). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out according to Hirabayashi (1981). After electrophoresis, gels were stained generally with CBB-R or with CBB-G (Neuhoff et al., 1988), if necessary. Immunoblotting was performed according to Towbin et al. (1979). After blocking with 2% BSA in TBS for 1 hr, the membrane was incubated with the rabbit primary antibody against chicken small intestinal mucosa TM (Xie et al., 1992) and subsequently with the biotinylated goat-anti rabbit IgG antibody. The antigen-antibody reaction was amplified with the ABC reagent (VECTASTAIN ABC kit, Vector Laboratory Inc.) and the immunoreactive proteins were detected using a Konica Immunostain Kit (Konica).
Polymerized rabbit skeletal muscle actin (1 mg/ml) and chicken liver TM (0.25 mg/ml) were mixed well in a solution consisting of 0.1 M KCl, 20 mM Tris-HCl, 2 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF and 0.1 mM DTT, pH 7.5 and incubated at 25°C for 2 hr. F-actin or TM alone was also incubated in the same way as a control. Then, the samples were ultracentrifuged at 250,000 × g for 20 min using a Beckman TLA 100.3 rotor. The resultant supernatants and the precipitates were analyzed by 2D-PAGE.
TM-affinity column chromatography
Purified chicken liver TM (2 mg) was coupled to 1 ml of HiTrap NHS-activated Sepharose 4B column (amersham pharmacia biotech) according to the manufacturer's protocol. The BSA-affinity column was prepared in the same way to detect nonspecifically bound proteins to the TM-affinity column. The liver proteins were extracted from the liver tissue or acetone powder with 20 volumes of the buffer consisting of 10% sucrose, 1 M KCl, 50 mM Tris-HCl, 20 mM EDTA, 0.1 mM PMSF and 0.1 mM DTT, pH 8.0 at 4°C for overnight. After centrifugation, the resultant supernatant was subjected to ammonium sulfate fractionation in 0–60% saturation. Centrifugal precipitates were dialyzed against the equilibration buffer containing 50 mM KCl, 20 mM Tris-HCl, 2 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF and 0.1 mM DTT, pH 7.5. Then, the proteins from the chicken liver tissue or acetone powder were applied onto the column. Purified GDH was also loaded on the TM-or the BSA-affinity column to ensure the association with TM. After the column was washed with 10 or 20 column volumes of the equilibration buffer, bound proteins were eluted with 15 column volumes of a linear concentration gradient of KCl from 50 mM to 1 M. Eluted fractions of 1.5 ml in each were collected and analyzed by SDS-PAGE.
Partial amino acid sequencing of the TM-binding protein
Effluents from the TM-affinity column were concentrated using Centricon-10 (Amicon) and subjected to 2D-PAGE. After staining the gel with CBB-R, the spot of the TM-binding protein was cut out and dialyzed against 62.5 mM Tris-HCl, 10% glycerol, 5% 2-mercaptoethanol and 1% SDS, pH 6.8 at 4°C for overnight. Then, the dialysate was treated with Staphylococcus aureus V8 protease as described previously (Cleveland et al., 1977). Protease-digested fragments were separated on SDS-PAGE and the gel was transferred to the Immobillon-P membrane. The CBB-R-stainable fragment was excised and the N-terminal sequence was determined with a 477A protein sequencer (Applied Biosystems). The obtained amino acid sequence was analyzed by homology search with SWISS-PLOT.
GDH enzymatic assay
Enzymatic activities of GDH were measured spectrophotometrically at 340 nm using a Beckman DU-530 spectrophotometer in the direction of reductive amination of 2-oxoglutarate into L-glutamate. The reaction solution was composed of 0.1 M ammonium acetate, 50 mM triethanolamine, 7 mM 2-oxoglutarate, 2.5 mM EDTA, 1 mM ADP and 0.2 mM NADH (Schmidt, 1974). One unit of enzyme was defined as the amount of enzyme required oxidizing 1 μM NADH per min at 25°C.
Purification of chicken liver TM
For detection of novel TM-binding proteins from nonmuscular sources by biochemical approaches, it is indispensable to obtain highly pure nonmuscle TM because of the possibility that misleading results might be caused by minor contaminants in preparations. Recently, we succeeded to purify the sea urchin egg TM of remarkably high purity by extracting TM from the agarose-IEF gels at the final purification step (Tobita et al., 1996). Considering that the amount of TM is far less in nonmuscular tissues than in muscular ones, we isolated nonmuscle TM from the chicken liver according to the purification procedure. As a results, we could prepare highly purified liver TM (Fig. 1), that was cross-reacted with the anti-TM antiserum (Fig. 1A, lane W). The 2D-PAGE pattern showed that the chicken liver TM was composed of multiple high and low Mr isoforms ranging from 32 to 45 kDa (Fig. 1B). About 2 mg of TM was obtained from 10 g of the chicken liver acetone powder. Since Graceffa and Lehrer revealed that the head-to-tail polymerization of TM is irreversibly lost in contact with 8 M urea for a long time (Graceffa and Lehrer, 1986), we carried out co-sedimentation assays of TM with purified rabbit skeletal muscle F-actin to examine whether the purified TM had its authentic nature (Fig. 2). While TM isoforms alone were scarcely precipitated, they were pelleted in the presence of F-actin, indicating that the purified proteins were genuine TM.
Identification of the nonmuscle TM-binding protein in the chicken liver
In order to find out novel nonmuscle TM-binding proteins from the chicken liver, we prepared the TM-affinity column according to the manufacturer's protocol and carried out column chromatography. We also made the BSA-affinity column to distinguish proteins nonspecifically bound to the TM-affinity column from specific ones. The total extract from the chicken liver acetone powder was loaded onto the columns (Fig. 3). After washing with 10 column volumes of the equilibration buffer, bound proteins were eluted with an increasing concentration of KCl. Two proteins of 57 and 45 kDa were mainly detected by SDS-PAGE in the effluents from the TM-affinity column (Fig. 3A). Although almost the same amounts of the 45 kDa protein were eluted from both columns (Fig. 3B), the 57 kDa protein was eluted exclusively from the TM-affinity column and scarcely from the BSA-affinity column. When the total extract from the fresh tissue was applied onto the TM-affinity column, the protein of 57 kDa was also eluted, although its amount was considerably smaller (data not shown).
To examine whether the 57 kDa protein is a novel TM-binding protein, the protease-digested fragment of the 57 kDa protein was sequenced and the resultant N-terminal amino acid sequence was analyzed by homology search with SWISS-PLOT. The result indicated that the obtained partial amino acid sequence was nearly identical to the N-terminal region of chicken liver glutamate dehydrogenase (GDH) (Moon et al., 1973), that has not been reported to bind to TM so far (Fig. 4).
GDH directly associates with liver TM
To ensure the interaction between TM and GDH, we isolated GDH from the chicken liver through several column chromatographic steps as described in Materials and Methods. Fractions obtained during the purification steps were run on SDS-PAGE (Fig. 5A) and their enzymatic activities were represented in Table I. We could purify chicken liver GDH more than 400-fold in the relative enzymatic activity and the result of gel filtration column chromatography at the final purification step revealed its molecular mass of approximately 330 kDa, presumably showing a formation of a native hexamer (data not shown). The purified GDH was also subjected to 2D-PAGE (Fig. 5B) and shown to have several isoforms with approximately the same molecular weight of 57 kDa and different isoelectric points, suggesting that the GDH isoforms were derived from the several distinct genes or the post-translational modifications. All the protein spots were detected by immunoblotting with the anti-GDH antibody (Rockland, Inc.) (data not shown).
Enzymatic activities during the purification steps of GDH.
In order to determine whether GDH indeed associates with TM, the purified GDH was applied onto the TM-affinity column and column chromatography was performed in the same way as in Fig. 3 with the exception that the column was washed with 20 column volumes of the equilibration buffer after sample loading. GDH was retained on the TM-affinity column (Fig. 6A), while scarcely on the BSA-affinity column (Fig. 6B), as judged by SDS-PAGE. These results indicated that GDH directly associated with nonmuscle TM.
It is known that GDH is involved in the pathway of glutamate metabolism. Previous biochemical analyses revealed that the enzymatic activity is influenced by nucleotides (Smith et al., 1975). In an attempt to address whether the interaction between TM and GDH is altered by nucleotides, we carried out affinity column chromatography in the presence of 0.1 mM ATP (Fig. 7B). As a consequence, the amount of GDH that bound to the TM-affinity column was greatly reduced in the presence of 0.1 mM ATP (Fig. 7B, lane t in E) relative to that in the absence of ATP (Fig. 7A, lane t in E), suggesting that the association of TM with GDH was regulated by ATP.
In the present study, we have demonstrated for the first time that chicken liver TM bound to GDH by affinity column chromatography and that the association of these two proteins was inhibited by ATP. Nevertheless, we found that not all GDH was retained on the TM-affinity column and the discernible amount of GDH was flowed through by washing with the equilibration buffer. It is conceivable that the affinity of TM to GDH might not be so high or that only the specific TM isoform(s) can bind to GDH, because purified chicken liver TM is composed of multiple high and low Mr isoforms. These possibilities remain to be investigated in the future study.
We have tried to confirm the association of TM with GDH by other procedures. Co-sedimentation assays of TM and GDH with F-actin were carried out, because it is strongly suggested that TM-binding protein can interact with TM in association with actin filaments, and because a small amount of actin was also eluted from the TM-affinity column (data not shown). However, a considerable amount of GDH was precipitated even in the absence of TM or F-actin, presumably owing to its tendency to aggregate (Smith et al., 1975), making it difficult to judge whether GDH bound to TM on the actin filaments or not. The gel overlay method using biotinylated TM could not give the reliable results either, because it was almost impossible to eliminate nonspecific signals from presumably endogenous biotinylated proteins in the chicken liver, although various conditions of blocking and washing were tested. Therefore, we concluded that the affinity column chromatography is the best way to show clearly the interactions between TM and other proteins in the liver.
GDH is a ubiquitous protein that is widely expressed in organisms from prokaryotes to eukaryotes and is involved in the pathways of glutamate metabolism, insulin release, ureagenesis, and so on. In vertebrates, it is generally accepted that GDH is localized in mitochondria (Smith et al., 1975). However, recent several investigations have indicated that GDH also exists in nuclei (Di Prisco and Garofano, 1975; McDaniel, 1995), lysosomes and/or vesicles (Rajas and Rousset, 1993; Rajas et al., 1996), and endoplasmic reticulum (Kawajiri et al., 1977; Lee et al., 1999). In addition, two novel GDH isoforms have been identified in the rat brain. There are some differences between them in the primary structure, heat stability, and kinetic parameter (Cho et al., 1995). Therefore, it is possible to say that the GDH isoforms from nonmitochondrial sources can be structurally and functionally distinguished from the mitochondrial one, although their physiological properties have not been understood in detail.
It has been reported that some enzymes interact with cytoskeletal proteins. For instance, the glycolytic enzyme, aldolase, is known to associate with the filamentous actin at low ionic strength (Arnold and Pette, 1968) and its binding site on actin has also been determined (O'Reilly and Clarke, 1993). Since colocalization of aldolase and F-actin has been observed in vivo (Pagliaro and Taylor, 1988), it is strongly suggested that the interaction between these two proteins is physiologically relevant. In addition, binding of aldolase with F-actin is inhibited in the presence of its substrate, fructose 1,6-bisphosphate, and affects the enzyme activity (Walsh et al., 1977). Thus, F-actin can work not only as a scaffold of the glycolytic enzyme but also as a regulator of the enzymatic activity. Our preliminary study showed that the enzyme activity of GDH was not affected significantly in the presence of TM (data not shown), but the more detailed study to see if TM influences on the GDH activity is underway.
A novel 58 kDa microtubule-associated protein has been found to be localized in the membrane of Golgi apparatus and Golgi-derived vesicles (Bloom and Brashear, 1989). Recently, it has been shown that the protein is identified as formiminotransferase cyclodeaminase that is involved in histidine metabolism (Bashour and Bloom, 1998; Hennig et al., 1998). It has been reported that the GDH isoform located at the lysosomal and/or vesicular membrane has also the capability to bind to microtubules in an ATP-dependent manner (Rajas and Rousset, 1993; Rajas et al., 1996). Although the physiological properties of these two enzymes in association with microtubules have not been elucidated, the activities of these emzymes might be somehow regulated by microtubules as in the case of aldolase by F-actin. Alternatively, these enzymes might function as a ligand for linking cytoskeletal structures to organelles. Considering that TM can be related to organelle and/or vesicle transport (Hegmann et al., 1989; Perham et al., 1996), the previous studies encourage us to propose that the interaction of TM with GDH may play significant roles in intracellular metabolisms.
We have reported for the first time that GDH is associated with TM by affinity column chromatography. Therefore, it seems interesting to examine more precisely the intracellular localization of GDH, because it is well known that TM is distributed in microfilaments and ruffling membranes (Lin et al., 1997), but not in mitochondria. In addition, our present study has shown that ATP influences the association of TM with GDH. Since the GDH activity is prevented in the presence of ATP at pH 7.5 and activated by ADP (Smith et al., 1975), it is suggested that ATP can regulate its binding of TM and the enzymatic activity. Future biochemical and immunocytochemical studies and molecular cloning can give further insights into the physiological role of the interaction between TM and GDH.
We would like to express our thanks to Dr. Osamu Numata (Institute of Biological Sciences, University of Tsukuba, Tsukuba, Japan) for generously allowing us to use a FPLC System (amersham pharmacia biotech) and to Mrs. Hiroko Kondou (Gene Experiment Center, University of Tsukuba, Tsukuba, Japan) for partial amino acid sequencing.