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T Takabe

Publications and source records attributed to T Takabe.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crystallization and preliminary X-ray studies of plastocyanin from Silene expressed in E. coli.

Plastocyanin from Silene (SilPc) expressed in E. coli has been crystallized in a form suitable for X-ray diffraction analysis by a macroseeding method using ammonium sulfate as a precipitant in acetate buffer (pH = 5.5). These crystals belong to the trigonal space group P3(1)21 or P3(2)21 with lattice parameters a = b = 76.6, c = 65.5 A, indicating an asymmetric unit containing two plastocyanin molecules. The crystals diffracted to at least 2.0 A.

Journal Article↗
PubMed1997

Isolation and characterization of a dnaK genomic locus in a halotolerant cyanobacterium Aphanothece halophytica.

We cloned and characterized a genomic locus encoding a distinct member of the DnaK/Hsp70 family of molecular chaperones, dnaK1, from the halotolerant cyanobacterium Aphanothece halophytica. Co-expression of dnaK1 with a plant plastocyanin precursor in Escherichia coli resulted in a dramatic increase in the solubility of the plant protein. This indicates that A. halophytica dnaK1 encodes a functional protein possessing functions assigned to DnaK/Hsp70 chaperone members. The A. halophytica dnaK1 locus also encompasses grpE and dnaJ homologue genes in the order grpE-dnaK1-dnaJ. The transcript content of dnaK1 increased strongly upon subjecting cyanobacterial cells to heat stress. Northern analyses using specific probes indicated transcript species of 2.8, 2.2, 1.3, and 0.7 kb, which comprised grpE-dnaK1, dnaK1, dnaJ, and grpE, respectively. This indicates the presence of different terminators and/or heat stress promoters in this locus. Both dnaK1 transcript and protein levels increased in cyanobacterial cells transferred to hyperosmotic environments, suggesting a role of DnaK1 in the protection and/or recovery of A. halophytica from this particular stress.

Amino Acid Sequence↗
PubMed1997

Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza sativa L.), albeit considered a typical non-glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salt-tolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C-terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno-gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt-inducible.

Aldehyde Oxidoreductases↗
PubMed1996

Kinetic and cross-linking studies on the interactions of negative patch mutant plastocyanin from Silene pratensis with photosystem I complexes from cyanobacteria, green algae, and plants.

The site-directed mutants of negative patches on silene plastocyanin (PC) were used to investigate the change of interactions between photosystem I (PSI) and PC during the course of evolution from cyanobacteria to plants. The net charges of two highly conserved negative patches (#42-45 and #59-61) on silene PC were systematically modified from -4 to +1. PSI complexes from cucumber and Chlamydomonas reinhardtii were efficient electron acceptors for silene PC. The increase of net charge on the negative patch (#42-45) of silene PC decreased the reduction rates of PSI from cucumber and Chlamydomonas, while the modification of the other negative patch (#59-61) had no effect. Though the addition of MgCl2 decreased the reduction rate of cucumber PSI, the decrease was severely diminished in the case of Chlamydomonas PSI, and the reduction rate increased with increasing concentration of MgCl2 when the net charge of the negative patch (#42-45) was modified to +1. The PSI complexes from Anabaena variabilis and Synechosystis sp. PCC 6803 were inefficient electron acceptors for silene PC and their rates were almost independent of the net charge of the negative patches, as well as the ionic strength of the reaction mixtures. Silene PC specifically cross-linked to the PsaF subunit of PSI complexes from cucumber, Chlamydomonas, Anabaena, and Synechosystis sp. PCC 6803. Modification of the negative patch (#42-45) inhibited the formation of cross-linked adducts in all the cases examined, whereas modification of the other negative patch (#59-61) had essentially no effect. Based on these results, the changes of electrostatic interactions between PC and PSI during the course of evolution from cyanobacteria to plants are discussed.

Amino Acid Sequence↗
PubMed1995

Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid.

When subjected to salt stress or drought, some vascular plants such as barley respond with an increased accumulation of the osmoprotectant glycine betaine (betaine), being the last step of betaine synthesis catalyzed by betaine aldehyde dehydrogenase (BADH). We report here cloning and characterization of BADH cDNA from barley, a monocot, and the expression pattern of a BADH transcript. An open reading frame of 1515 bp encoded a protein which showed high homology to BADH enzymes present in other plants (spinach and sugar-beet) and in Escherichia coli. Transgenic tobacco plants harboring the clone expressed high levels of both BADH protein and its enzymatic activity. Northern blot analyses indicated that BADH mRNA levels increased almost 8-fold and 2-fold, respectively, in leaves and roots of barley plants grown in high-salt conditions, and that these levels decreased upon release of the stress, whereas they did not decrease under continuous salt stress. BADH transcripts also accumulate in response to water stress or drought, indicating a common response of the plant to osmotic changes that affect its water status. The addition of abscisic acid (ABA) to plants during growth also increased the levels of BADH transcripts dramatically, although the response was delayed when compared to that found for salt-stressed plants. Removal of plant roots before transferring the plants to high-salt conditions reduced only slightly the accumulation of BADH transcripts in the leaves.

Abscisic Acid↗
PubMed1995

Expression and characterization of Met92Gln mutant plastocyanin from Silene pratensis.

To investigate the role of the copper-ligand Met92 in the structural and functional properties of silene plastocyanin (PC), Met92 was replaced with Gln, which is the purposed fourth copper-ligand in another blue copper protein, stellacyanin. By use of the recently developed expression system [Hibino et al. (1994) J. Biochem. 116, 826-832], the Met92Gln mutant of intermediate precursor plastocyanin was successfully expressed in Escherichia coli and accumulated in the periplasmic space as a mature protein. In contrast to the wild type, most of the Met92Gln mutant PC accumulated as an apoprotein. After purification, mutant apoprotein could incorporate copper ions, although less efficiently than the wild-type apoprotein. The absorption peak of Met92Gln mutant PC was blue-shifted from 597 nm in the wild type to 591 nm. The rhombic type EPR spectrum was obtained for the mutant in place of the axial spectrum in the wild type. Compared with that of the wild-type PC, the oxidation-reduction potentials of the Met92Gln mutant PC were lower by about 35 mV over the whole pH range examined. These results indicate that the Met92Gln mutant exhibited "stellacyanin-like" spectroscopic properties. Interestingly, the electron-transfer activities of the mutant PC with the physiological electron donor (cytochrome f) and acceptor (Photosystem I) were similar to that of the wild-type PC. Since Met92 is conserved in all the plastocyanins whose primary structures are known, we propose that the primarily function of Met as a copper-ligand is in the uptake of copper ions during folding rather than in electron-transfer activities.

Base Sequence↗
PubMed1995

Site-directed mutagenetic study on the role of negative patches on silene plastocyanin in the interactions with cytochrome f and photosystem I.

To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Molecular cloning of cDNA for a 17.5-kDa polypeptide, the psaL gene product, associated with cucumber Photosystem I.

Three cDNA clones encoding the 17.5-kDa polypeptide of cucumber Photosystem I were isolated and sequenced. A cDNA insert of them was 812 bp long and contained a long open reading frame that encoded a polypeptide with 217 amino acid residues. The deduced amino acid sequence of the cDNA was 77% and 71% identical to the amino acid sequences of psaL gene products from barley and spinach, respectively.

Amino Acid Sequence↗
PubMed1994

Role of transit peptide sequence of plastocyanin for its expression, processing, and copper-binding activity in Escherichia coli.

Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed. Their expression, processing, and copper-binding activity have been examined. When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E. coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates. Its accumulation level was increased by the addition of copper ions. About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein. On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size. The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions. The mature plastocyanin content corresponds to 8% (w/w) of the total E. coli protein content (123 mg per liter of culture). The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin. Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Characterization of genes that encode subunits of cucumber PS I complex by N-terminal sequencing.

N-terminal amino acid sequencing was carried out to characterize the genes of the cucumber PS I complex (PSI-100) that contains eight polypeptides and catalyzes the light-dependent transfer of electrons from plastocyanin to ferredoxin. The genes of all subunits except the 17.5 kDa polypeptide in PSI-100 have been identified. These are psaA/psaB (65/63 kDa), psaD (20 kDa), psaE (19.5 kDa), psaF (18.5 kDa), psaH (7.6 kDa), and psaC (5.8 kDa). The 17.5 kDa polypeptide is a new protein and is designated tentatively as the gene product of psaM. N-terminal amino-acid sequencing indicated the presence of two polypeptides in the 7.6 kDa band. One of these is the gene product of psaH and is essential for the activity of the PS I complex, and the other one is as yet unrecognized and largely depleted in the PSI-100 complex. Gene products of psaG, psaI, and psaK, which have been proposed as the components of PS I complex, are not involved in the PSI-100 complex, but are involved in the PS I complex (PSI-200), which contains 120 chlorophyll per reaction center chlorophyll (P700) and light-harvesting chlorophyll a/b protein complexes. Three polypeptides (26,23 and 22.5 kDa) are not involved in the PSI-100 and are assigned as the apo-protein of light-harvesting chlorophyll a/b protein complexes.

Amino Acid Sequence↗
PubMed1991

Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli.

The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein. The purified precursor apo-plastocyanin was processed to the mature apo-plastocyanin by chloroplast extracts. N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature plastocyanin that was deduced from the nucleotide sequence. The copper could be incorporated into the apo-plastocyanin of mature size in vitro, but could not into the precursor apo-plastocyanin under the same conditions. Absorption spectra and reduction potential of the reconstituted mature plastocyanin were indistinguishable from those of the purified spinach plastocyanin. The electron transfer activities of the reconstituted plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach plastocyanin.

Amino Acid Sequence↗
PubMed1991

Sequence and expression of genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chromatium vinosum.

A DNA fragment bearing genes for the large (rbcL) and small (rbcS) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was cloned from the photosynthetic purple sulfur bacterium Chromatium vinosum. Enzymatically fully active RuBisCO was synthesized in Escherichia coli cells when the cloned DNA was placed downstream of tac promoter. Nucleotide (nt) sequences of rbcL-rbcS were more homologous to cyanobacterial counterparts than to those from Alcaligenes eutrophus or higher plants. However, the amino acid (aa) sequence in a domain responsible for CO2 activation in the C. vinosum rbcL product resembled the corresponding aa sequence in higher plant RuBisCos, but not in the cyanobacterial enzymes. Chemically determined aa sequences at the N terminals of both subunits of RuBisCO purified from C. vinosum were not identical to those deduced from the nt sequences, although they were completely the same as aa sequences deduced from rbcA-rbcB, another locus encoding RuBisCO in C. vinosum. Therefore, the rbcL-rbcS locus seems to be barely expressed under a standard condition for photoautotrophic growth. The homology of the nt sequences between rbcL and rbcA was 82%, and that between rbcS and rbcB was 63%, whereas the codon usages of these genes were basically identical. The rbcL-rbcS and rbcA-rbcB loci therefore must have evolved from a common ancestral set of genes after duplication, instead of lateral gene transfer.

Amino Acid Sequence↗
PubMed1991

Subunit composition of Photosystem I complex that catalyzes light-dependent transfer of electrons from plastocyanin to ferredoxin.

The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.

Darkness↗
PubMed1989

Kinetic studies on a cross-linked complex between plastocyanin cytochrome f.

A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.

Carbodiimides↗
PubMed1989

Immunochemical Analysis Shows That an ATP/ADP-Translocator Is Associated with the Inner-Envelope Membranes of Amyloplasts from Acer pseudoplatanus L.

Pure preparations of intact amyloplasts and chloroplasts, free from mitochondrial contamination, were isolated from cultured cells of the white-wild and green-mutant lines of sycamore (Acer pseudoplatanus L.), respectively. A specific rabbit antiserum against yeast mitochondrial cytochrome c(1) only cross-reacted with mitochondrial membranes from the white-wild sycamore cells. The outer and inner envelope-membranes of the two plastid-types were isolated and subsequently analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to characterize polypeptide patterns in each fraction. Analysis by immunoblotting clearly showed that antiserum against the 29-kilodalton inorganic orthophosphate translocator isolated from pea chloroplasts cross-reacted with a 31-kilodalton polypeptide residing in the inner-envelope membranes from both sycamore chloroplasts and amyloplasts. In contrast, antiserum against the ADP/ATP-translocator isolated from mitochondria of Neurospora crassa yielded a positive signal with a 32-kilodalton polypeptide in the inner-membranes isolated from amyloplasts, but not green-mutant chloroplasts. We propose that this 32-kilodalton polypeptide in the amyloplast envelope is a putative ATP/ADP-translocator and its possible functional significance is discussed.

Journal Article↗
PubMed1988

Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.).

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.

Journal Article↗