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T Takabe

Publications and source records attributed to T Takabe.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg(2+)-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg(2+), Mn(2+) and Ca(2+), whereas it was not affected by Sr(2+), K(+), or Na(+). The K(m) for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N'-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg(2+)-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F(1) ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).

Journal Article↗
PubMed1988

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities.

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO(2) fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.

Journal Article↗
PubMed1987

Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody.

Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatography. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit.

Aldehyde Oxidoreductases↗
PubMed1986

Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition.

A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.

Centrifugation, Density Gradient↗
PubMed1986

Role of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in the activation process.

The large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from the cyanobacterium Aphanothece halophytica and from the purple sulfur photosynthetic bacterium Chromatium vinosum (strain D) were separated by sucrose density gradient centrifugation at low ionic strength and alkaline pH (9.3), respectively. It was found that subunit B enhances the extent of activation by CO2 and Mg2+ at equilibrium of the two homologous enzymes consisting of Aphanothece large subunit and its own small subunit (AaBa) and the Chromatium large subunit and its own small subunit (AcBc). The extent of activation induced by saturating amounts of subunit B was larger with AcBc than AaBa, amounting to 3.7- and 1.8-fold of that by each catalytic core alone, respectively. Subunit B stimulated both the extent of activation at equilibrium and catalysis in a parallel and simultaneous manner with respect to the concentration of B in both homologous enzymes. These results suggest that subunit B interacts with both activation and catalytic sites simultaneously. On the other hand, Chromatium subunit B only slightly stimulated the extent of activation in the hybrid enzyme AaBc. The role of subunit B in enhancing the extent of activation at equilibrium can be substituted by the effect exerted by 6-phosphogluconate. Both homologous enzymes AaBa and AcBc showed a faster deactivation rate when the enzyme was activated in the absence of subunit B. The mechanism by which subunit B promotes activation seems to involve its effect on stabilizing the activated enzyme molecule. From studies on the Km for substrate CO2 in the hybrid enzyme AaBc a major involvement of subunit B in influencing Km (CO2) seems unlikely.

Carbon Dioxide↗
PubMed1986

Charges on proteins and distances of electron transfer in metalloprotein redox reactions.

A modified form of the Debye-Marcus equation relating electron transfer rate constants to charges on proteins and distances of electron transfer has been applied to the reaction of chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups. The rate of electron transfer from reduced cytochrome f to ferricyanide decreased with increasing ionic strength when the native and singly substituted cytochrome f were used, although a sharp decrease was observed in the former case. When doubly or more than triply substituted cytochrome f was used, the rate of electron transfer was almost constant or increased with increasing ionic strength, respectively. The kinetic-ionic strength effects on this reaction can be well explained by the Debye-Marcus equation in which the charge and radius of the protein are treated as variable parameters. The results show the importance of local positive charges of about 2.0 on native cytochrome f and effective radius of about 11 A of cytochrome f for the electron transfer to ferricyanide. Since the net charge on the native cytochrome f is negative and the calculated radius of the protein is 22.8 A, the above results indicate that positive charges on the electron transfer site control the electrostatic interactions in this reaction. Previously reported data which had been analyzed by using the total net charge and full radius of the protein, were also well explained by the local charge and effective radius of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochromes↗
PubMed1986

Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex.

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b(6)/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b(6), and Rieske Fe-S center protein in cytochrome b(6)/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b(6)/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b(6), and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.

Journal Article↗
PubMed1986

Effect of Betaine on Enzyme Activity and Subunit Interaction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Aphanothece halophytica.

The presence of betaine, a quaternary ammonium compound, at a concentration (0.5 molar) reported to accumulate inside Aphanothece halophytica in response to increasing external salinity, slightly promoted ribulose-1,5-bisphosphate (RuBP) carboxylase activity. KCl at 0.25 molar inhibited RuBP carboxylase about 55%. Betaine relieved the inhibition by 0.25 m KCl and the original uninhibited activity was restored at 1 m betaine. Other osmoregulatory solutes such as sucrose and glycerol also reduced KCl inhibition, though to a lesser extent than betaine. Proline had no effect. The protective effect of betaine against KCl inhibition of RuBP carboxylase activity was also observed in other cyanobacteria, i.e. Synechococcus ACMM 323, Plectonema boryanum, and Anabaena variabilis, and in the photosynthetic bacterium Rhodospirillum rubrum but not in Chromatium vinosum. Apart from betaine, other quaternary ammonium compounds, i.e. sarcosine and trimethylamine-N-oxide (TMAO), but not glycine, also protected the enzyme against KCl inhibition and the effectiveness of such compounds appeared to correlate with the extent of N-methylation. Heat and cold inactivation of the enzyme could be protected by either betaine or KCl. However, best protection occurred when both betaine and KCl were present together. The K(m) (CO(2)) was not altered by either betaine or KCl, nor when they were present together. However, the K(m) (RuBP) was increased about 5-fold by KCl, but was unaffected by betaine. The presence of betaine together with KCl lowered the KCl-raised K(m) (RuBP) by about half. The extent of the dissociation of the enzyme molecule under the condition of low ionic strength was reduced by either betaine or KCl alone and more so when they were present together. Glycine, sarcosine, and TMAO were more effective than betaine or KCl in lowering the extent of the dissociation of the enzyme molecule.

Journal Article↗
PubMed1985

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli.

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.

Chromatium↗
PubMed1985

Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities.

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.

Catalysis↗
PubMed1985

Factors affecting the dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase/oxygenase from Aphanothece halophytica.

Factors affecting the mutual interaction between the catalytic core [octamer of large subunit (A)] and the small subunit (B) comprising ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from the superhalophilic cyanobacterium, Aphanothece halophytica, were investigated. The enzyme molecule dissociated into the catalytic core highly depleted of subunit B and the monomeric form of subunit B during density gradient centrifugation (15 h, 4 degrees C) in a sucrose solution of low ionic strength ([I] less than or equal to 50 mM), whereas dissociation was effectively prevented in the presence of 0.3 M KCl. Under the latter condition, dissociation of the enzyme molecule was almost completely prevented by raising the temperature to 20 degrees C, suggesting hydrophobic interaction between catalytic core and subunit B. The addition of RuBP to the sucrose gradient was shown to effectively reduce the molecular dissociation, suggesting a close interaction between the catalytic site and the binding site of subunit B with the catalytic core directly or indirectly. The dissociation was accelerated at alkaline pH higher than 8.5. Reconstitution of the enzymatically active molecular form from the separated components, catalytic core highly depleted of subunit B and B1, was done under various conditions. Both carboxylase and oxygenase activities increased proportionately with the amount of subunit B and then became saturated. From the reconstitution kinetics of RuBP carboxylase, the binding constant of subunit B (KD) was estimated to be about 30 nM in the presence of bovine serum albumin under the usual assay conditions at pH 7.5 and 25 degrees C, but decreased to about 1 nM by the further addition of 0.3 M KCl. Alkaline pH (8.5 or 9) could increase KD by one order of magnitude. High KD was also observed as a result of lowering the temperature; however, the presence of 0.3 M KCl or 0.4 M sucrose or glycerol could effectively decrease the KD at low temperature from 900 nM to less than 50 nM. All these data indicate that the enzyme dissociation at low temperature can be prevented in vivo by cellular components such as salts, polyols, and substrate RuBP besides a factor of enzyme concentration.

Catalysis↗
PubMed1984

Essentiality of the small subunit (B) in the catalysis of RuBP carboxylase/oxygenase is not related to substrate-binding in the large subunit (A).

The small subunit (B) of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase from Aphanothece halophytica is absolutely required for the catalysis, but depletion of subunit B does not significantly affect the formation of the quaternary complex-[enzyme.activator CO2.Mg.carboxyarabinitol bisphosphate] in the catalytic core. The inhibition of RuBP carboxylase activity by the reaction of the epsilon-amino group of a lysine in the RuBP-binding site with pyridoxal 5-P is the same whether subunit B is added to the catalytic core before or after the inactivating reaction. The function of subunit B is not related to the substrate binding.

Binding Sites↗
PubMed1984

Interaction of constituent subunits in ribulose 1,5-bisphosphate carboxylase from Aphanothece halophytica.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) from the halophilic cyanobacterium, Aphanothece halophytica, dissociates into catalytic core (large subunit A oligomer) and small subunit B under low ionic strength during sucrose density gradient centrifugation. Supplementation of KCl, NaCl, or K2SO4 ( [I] = 0.3 M) partly prevents the dissociation, the preventive effect of divalent cation salts such as MgCl2 and CaCl2 being more effective than monovalent cation salts. RuBisCO with its higher-plant-type molecular form can be isolated from the cyanobacterial extracts using gradient medium containing 0.3 M KCl, 20 mM MgCl2, and 10 mM CaCl2. The isolated enzyme contains large subunit A and small subunit B in a molar ratio of approximately 1:1, estimated from the densitometric scanning of Coomassie blue-stained gels. During the second sucrose density gradient centrifugation to remove minor contaminants, a small amount of subunit B is depleted from the holoenzyme. Determination of the molecular weight by equilibrium centrifugation and electron microscopic observation have confirmed that the cyanobacterial RuBisCO has an A8B8-type structure. The enzyme activity per se is found to be sensitive to concentrations of salts, and small subunit B is obligatory for the enzyme catalysis. It has been shown that the more the enzyme activity is inhibited by salts, the tighter the association of subunit B becomes. It is likely that the active enzyme retains the loose conformational structure to such an extent that the dissociable release of subunit B from the holoenzyme in vivo is not allowed.

Calcium Chloride↗
PubMed1984

Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges.

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗
PubMed1984

Importance of local positive charges on cytochrome f for electron transfer to plastocyanin and potassium ferricyanide.

To study the relationship between the electron transfer rate and the net or local charge of protein, chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups, has been prepared by using an arylating reagent 4-chloro-3,5-dinitrobenzoic acid. Four distinct species of chemically modified cytochrome f, having 1 to 4 mol of modified amino residues per mol of cytochrome f, were separated by preparative polyacrylamide gel electrophoresis. The rate of electron transfer from the reduced singly substituted cytochrome f to the oxidized spinach plastocyanin was only about 50% of that of the native unmodified cytochrome f. The reaction rate further decreased about 50% upon the modification of each amino residue. The biphasic oxidation of cytochrome f by plastocyanin was observed when more than 2 mol of amino residues were modified. The rate of the second phase also decreased with an increasing number of modified amino residues. On the other hand, the oxidation of chemically modified cytochrome f by potassium ferricyanide was clearly monotonic. The rate decreased about 30% upon the modification of each amino residue. The midpoint potentials of chemically modified cytochrome f were almost the same as that of the native protein. These results clearly indicate the importance of local positive charges on cytochrome f, since the overall net charge of cytochrome f is negative at neutral pH. The theory of electrostatic corrected outer-sphere electron transfer of Marcus explained the effect of charge on cytochrome f for the reaction with the small molecule of ferricyanide well, but not the reaction with the protein of plastocyanin.

Cytochromes↗