Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca2 -induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca2 of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca2 causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca2 -binding protein called spasmin could not bind with Ca2 after TNM treatment, even if the treatment was performed in the presence of Ca2 . In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl-coumarinyl-maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca2 when spasmin binds with its receptor protein in the spasmoneme.
Species of protozoa belonging to the Vorticellidae ciliates, such as Vorticella, Carchesium and Zoothamnium, possess a unique contractile system that is independent of ATP or other organic fuels (Hoffmann-Berling, 1958; Amos et al., 1975; Asai et al., 1978). The bundle of 3-nm-diameter contractile filaments in the stalk is named the spasmoneme. The major component of the spasmoneme is spasmin, an EF-hand calcium binding protein. Another protein in the spasmoneme related directly or indirectly to the Ca2+-induced contraction is the putative spasmin-receptor protein (Asai et al., 1978). The contraction/extension of the spasmoneme can be repeated many times in vitro by the addition/removal of Ca2+ (Amos et al., 1975; Asai et al., 1978). Amos et al. (1975) suggested that spasmoneme contraction was due entirely to the spasmin molecule. However, mach indirect evidence has emerged that contradicts Amos' proposal (Yamada et al, 1985; Kono et al, 1997; Moriyama et al, 1999). Moriyama et al (1999) have revealed that the contraction is due to the molecular folding of spasmin and a putative spasmin - receptor protein. Our knowledge is still limited about the protein system performing the contraction. It is therefore worth verifying by a chemical method whether some amino acid residue(s) in a spasmoneme protein is essential for the contraction and whether the putative spasmin - receptor protein protects the active site of spasmin.
The chemical modification of amino acid residues with a special reagent is a good way to identify active sites in a protein. There are only a few reports on the amino acids essential for spasmoneme contraction (Kono et al., 1997; Fang et al. 2003). Tetraitromethane (TNM) is often used to investigate the role of tyrosine residues in protein (Nakata et al., 1986; Koffman et al., 1991; Beckingham et al., 2001). Fig. 1 shows the reaction with TNM. The rate of the reaction between TNM and the tyrosine residues in a protein increases as the pH is raised from 7.0 to 10.0. Nitration does not occur below pH 7.0 (Femfert et al., 1972; Koffman et al., 1991). In addition, TNM will decompose as the pH is raised (Sokolovsky et al., 1966). So the most suitable pH is 8.0–9.0. The present study provides insight into the importance of tyrosine residues in the spasmoneme. It is also the first to report the tyrosine residues essential for spasmoneme contraction.
MATERIALS AND METHODS
TNM and DACM were purchased from Sigma Chemical Co., St. Louis. The solutions used for the chemical modification of TNM contained 4 mM EGTA-2Na, 0.1 M KCl, 20mM bufferincluding sodium phosphate at pH 6.0–7.5, Sodium borate at pH 8.0–9.0 and glycine at pH 9.5–10.0 and TNM (0∼200 μM). The pH was adjusted according to different experiments. The DACM solution was freshly prepared in acetone and stored on ice in the dark (Ohyashiki et al., 1994). It contained 4 mM EGTA-2Na, 0.1 M KCl, 20 mM imidazole and DACM (0∼ 500 μM). The pH was controlled at pH 7.0.
Cell Culture and Glycerol Treatment
Vorticella sp. was found and collected at the Tianjin Jizhuanzi Sewage Facility, China. Fig. 2 shows the contraction and extension of glycerinated stalks on addition and removal of Ca2+.
The collected colonies were cultured in large low-form flasks containing 0.25% vita-shrimp/cereal leaves infusion at 20°C (Ochiai et al., 1979; Vacchiano et al., 1991). Cover glasses were put on the culture liquid surface. A few days later, a suitable number of zooids would attach to the glass. Each cover slip with cultured Vorticella cells was treated in a solution consisting of 0.1% saponins, 4 mM EGTA-2Na, 0.1 M KCl, and 20 mM imidazole, at pH 6.8 and 0°C, for 45 min. Then, the cells were transferred to a new solution containing 35% glycerol, 4 mM EGTA-2Na, 0.1 M KCl, and 20 mM imidazole, at pH 6.8 and 0°C, for 60 min.
Procedure for the Chemical Modification of TNM
Before the chemical modification experiment, the cover glasses were washed (Washing Solution: 4 mM EGTA-2Na, 0.1 M KCl, and 20 mM imidazole, pH 6.8) for 20 min in order to remove the glycerol on the surface of the stalks. The stalks were incubated with contraction solution (2 mM Ca2+, 0.1 M KCl, and 20mM imidazole, pH 6.8) for 10 min. Then, the cover glasses were photographed for counting the numbers of coils in stalks without TNM treatment and transferred to washing solution for 20 min in order to remove Ca2+. After these experiments, the cover glasses with stalks were incubated in petri dishes containing the chemical modification solution under various conditions. Then, the stalks still attached to the cover glasses were washed with washing solution for 20 min in order to remove TNM and transferred to the contraction solution. After these experiments, the stalks were photographed again.
Experimental Procedure for Examining the Effect of DACM on Vorticella Stalks
The procedure used was the same as that for the chemical modification with TNM. The cover glasses with stalks were incubated in solutions of 100∼500 μM DACM at 0°C and pH 7.0 for 60 min. In other experiments, the cover glasses with stalks were incubated in a solution of 500 μM DACM at 0°C, pH 7.0 for different periods of time.
The average numbers of coils in modified and unmodified stalks were estimated by counting the total for 100 individual stalks using the photos. Contractibility for modified stalks was taken as the ratio of the average number of coils in modified stalks to that in unmodified stalks. Namely,
Ca2+ Protection of Spasmoneme Contractibility against TNM
To investigate if preincubation with Ca2+ can protect the spasmoneme from denaturation, glycerinated stalks were pre-incubated with the contraction solution for 10 min. In control experiments, glycerinated stalks were not treated with the contraction solution before the TNM treatment. The stalks were modified with 20, 50 and 80 μM TNM solutions at pH 8.0 and 0°C for 60 min. The TNM was then removed by washing extensively. Next, the stalks were incubated with the contraction solution for 10 min and the coils were counted. Average numbers of coils in 100 stalks with and without pre-incubation with Ca2+ were calculated for determining contractibility.
Effect of TNM on Affinity of Spasmin for Ca2+
The method used to purify spasmin was based on previous research (Fang et al., 2003). The spasmin fraction in the absence of Ca2+ was modified with 100 μM TNM at 0°C for 60 min. Then, it was dialyzed against buffer (1 mM EGTA and 50 mM Tris-HCl, pH 7.5) and divided into two equal volumes and concentrated. 2 mM Ca2+ was added to one portion and 2 mM EGTA to the other. The spasmin fraction pre-incubated with Ca2+ was treated as described above. Native spasmins not treated with TNM, as controls, were incubated with 2 mM Ca2+ or 2 mM EGTA-2Na. These samples were resolved by 12.5% native polyacrylamide gel electrophoresis (Native-PAGE) (Ueno et al., 1999). In this experiment, the TNM solution lacked EGTA-2Na.
Effect of TNM Concentration on Stalk Contraction
The effect of TNM concentration on the spasmoneme was investigated. The ability of Vorticella stalks to contract was examined at various TNM concentrations (Fig. 3). As shown in Fig. 3, the contractibility of the stalks decreased about 90% as the TNM concentration rose from 0 to 100 μM. The contraction was evidently inhibited when the TNM concentration was 80–100 μM.
Effect of TNM on Stalk Contraction at Various pH
The effect of TNM on contraction was examined at various pH. In consideration of the decomposition of TNM with rising pH, we used 200 μM of TNM, although 100 μM obviously inhibited spasmoneme contraction at 0°C, pH 8.0.
The contractibility of spasmonemes not treated with TNM was unaffected at pH 6.0 to 8.5, which suggests that the loss in Fig. 4 was caused by the chemical modification.
Effect of DACM Modification on Vorticella Stalks
As expected, the contractibility of the stalks was unaffected by the various concentrations of DACM (Fig. 5A). Therefore, experiments with different treatment times were performed. The results showed that the period of treatment with DACM had no effect on contraction, either (Fig. 5B). So, it was concluded that the modification of cysteine residues does not affect spasmoneme contraction in Vorticella sp.
Ca2+ Prevents Loss of Spasmoneme Contractibility
Preincubation with Ca2+ protected the spasmoneme against the loss of contractibility caused by the chemical modification with TNM (Fig. 6).
Effect of TNM on the Ability of Spasmin to Bind Ca2+
Spasmin is the major component of spasmoneme and a Ca2+-binding protein. Native spasmin has been shown to exhibit a Ca2+-dependent shift on native PAGE (Yamada et al., 1982). In order to identify potential sites of the essential tyrosine residues in the spasmoneme, the effect of Ca2+ on spasmin was investigated. TNM-modified spasmin in the presence of Ca2+(Fig. 7A Lane 3) migrated at the same rate as without Ca2+(Fig. 7A Lane 4). The results of native PAGE of the chemically modified spasmin with and without Ca2+ implied that the affinity of spasmin for Ca2+ was reduced by TNM (Fig. 7A), which suggested that the loss of contractibility was due to the modification of spasmin. In Fig. 7B, pre-incubation with Ca2+ could not preserve the ability of spasmin to bind Ca2+.
TNM has been shown to be a convenient reagent for the nitration of tyrosyl residues in proteins at pH 8.0 (Sokolovsky et al., 1966; Riordan et al., 1966), although specificity studies have shown that cysteine residues are also potentially reactive towards TNM (Riordan and Christen, 1968). In the present study, it was revealed that spasmoneme contraction depended on the TNM concentration. The contractibility decreased nearly 90% with a rise in the TNM concentration from 0 to 100 μM at 0°C, pH 8.0. It is generally recognized that TNM oxidizes thiol groups at pH of 6.0 to 9.0 (Sokolovsky et al., 1966). So it was necessary to explore the effect of TNM on spasmoneme contraction at various pH and the function of cysteine residues in order to confirm the role of these residues in the contraction. The spasmoneme contraction was unaffected at pH 6.0–8.5 and 0°C (Fig. 4), which indicated that the loss of contractibility was caused by the chemical modification. DACM is a highly specific fluorescent thiol reagent, often employed to determine the role of cysteine residues in a protein's function (Tezuka et al., 1978; Ida and Tokushige, 1985; Werneburg and Ash, 1993). DACM treatments had proved that the chemical modification of cysteine residues did not affect the contraction of Vorticella (Fig. 5). In Ca2+ protection experiment, the contraction was not affected by TNM treatment when the spasmoneme was incubated with Ca2+ before the chemical modification (Fig. 6). These results suggest that tyrosine residues are essential for spasmoneme contraction.
Spasmin is the main component of the spasmoneme and spasmoneme contraction is induced by Ca2+ binding. Therefore, the spasmoneme may have lost the ability to contraction because spasmin cannot bind with Ca2+ after TNM treatment. Maciejewski et al. has proved that native spasmin bound to Ca2+ migrates at a slower rate than that free from Ca2+ on native PAGE (Maciejewski et al., 1999). We investigated the ability of spasmin to bind Ca2+ by native PAGE. The results (Fig. 7) showed that tyrosine residues contributed to the binding (Fig. 7A) and preincubation with Ca2+ could not preserve the ability of spasmin to bind Ca2+ (Fig. 7B).
In the present study, the result that preincubation with Ca2+ did not preserve the affinity of spasmin for Ca2+ (Fig. 7) seemed to contradict the finding that the preincubation prevented a loss of contractibility caused by TNM (Fig. 6). The discrepancy could have two explanations. One possibility is that spasmin can be modified by TNM because it undergoes a change in structure when isolated from the spasmoneme. But the isolated spasmin not treated with TNM still bound with Ca2+ (Fig. 7, lane 1 and 2), which implied that the spasmin structure was unchanged. So, this possibility can be excluded. On the other hand, Yamada et al showed that there was another protein, namely the putative receptor protein, which played a complementary role in spasmoneme contraction (Yamada et al, 1982). In addition, the results of chemical modifications with tryptophan (Kono et al, 1997) and histidine (Fang et al, 2003) confirmed that the receptor protein was essential for spasmoneme contraction. So, the second possibility is that the receptor protein plays a complementary role in protecting tyrosine residues from TNM in the spasmoneme. If this hypothesis can be confirmed, our observations on tyrosine residues modification might be a good starting point for the investigation of spasmoneme contraction.
This research was supported by grants from the Yamada Science Foundation (H.A.). We greatly appreciate the help, comments and suggestions provided by Dr. T. Itabashi and Dr. S.J. Jia. F.J. is a 21st Century COE research assistant of physics self-organization systems composed of multi-components.