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T Akazawa

Publications and source records attributed to T Akazawa.

At least 91 records · Page 5Linked to original sources
PubMed1988

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development.

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

Journal Article↗
PubMed1987

Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins.

Amyloplasts isolated from white-wild suspension-cultured cells of sycamore (Acer pseudoplatanus L.) are found to import and process the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach, but they lack the ability to form its holoenzyme due to the absence of both the large subunit and its binding-protein. They also import the precursor of the 33-kDa extrinsic protein (p33-kDa) of the O2-evolving complex of Photosystem II from spinach, but process is only to an intermediate form (i33-kDa). Chloroplasts from green-mutant cells of sycamore process p33-kDa to its mature form in this heterologous system. These results suggest that the thylakoid-associated protease responsible for the second processing step of p33-kDa is missing in amyloplasts, possibly due to the absence of thylakoid-membranes. In contrast, the apparent import of the precursor of the light-harvesting chlorophyll a/b-binding apoprotein (pLHCP) from spinach was not detected. Sycamore amyloplasts may lack the ability to import this particular thylakoid-protein, or rapidly degrade the imported molecules in the absence of thylakoid-membranes for their proper insertion.

Chlorophyll↗
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↗
PubMed1987

[Golgi body].

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Cell Fractionation↗
PubMed1987

[Amyloplast].

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Cell Fractionation↗
PubMed1986

Purification and characterization of heme-containing low-activity form of catalase from greening pumpkin cotyledons.

In germinating pumpkin seeds, catalase is synthesized as a precursor (59-kDa) form, with molecular mass larger than the mature molecule (55 kDa). Both the precursor and mature forms of catalase are localized in the microbodies, i.e., glyoxysomes and leaf peroxisomes [Proc. Natl. Acad. Sci. USA 81, 4809-4813 (1984)]. We have now purified the 59-kDa catalase precursor and compared its properties with those of the 55-kDa mature molecule. The molar catalytic activity of the 59-kDa catalase was tenfold lower than that of the 55-kDa molecule, whereas the heme content was found to be same, with both forms containing four hematin groups per molecule. It is inferred from these results that the low activity of the 59-kDa molecule is not related to the binding of heme to the protein, but presumably involves conformational differences between the 59-kDa and 55-kDa molecules. We have further found that the reduction of total catalase activity in pumpkin cotyledons during greening was due to a decrease in the amount of the enzymically active 55-kDa catalase accompanying an increase in the 59-kDa molecule.

Catalase↗
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

Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

Endoplasmic Reticulum↗
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

Protein Phosphorylation in Amyloplasts Isolated from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Highly purified amyloplasts were isolated from cultured cells of sycamore (Acer pseudoplatanus L.). Incubation of amyloplasts with [gamma-(32)P]-ATP resulted in the labeling of more than ten polypeptides. Pulsechase experiments showed the reversibility of the process with some but not all of the polypeptides. The phosphorylation reaction of one polypeptide, M(r) 100, was shown to be calcium dependent. Although exogenously added pig brain calmodulin had no effect, the calmodulin antagonist W-7 strongly inhibited phosphorylation of the 100 kilodaltons polypeptide. The presence of endogenous calmodulin, about 1 to 3 micrograms per milligram protein, in the amyloplast preparation was estimated by activation of phosphodiesterase in vitro.

Journal Article↗
PubMed1986

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F(0) to F(1) ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N'-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.

Journal Article↗
PubMed1986

Immunocytochemical Analysis Shows that Glyoxysomes Are Directly Transformed to Leaf Peroxisomes during Greening of Pumpkin Cotyledons.

The functional transition of glyoxysomes to leaf peroxisomes occurs during greening of germinating pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). The immunocytochemical protein A-gold method was employed in the analysis of the transition using glyoxysomal specific citrate synthase immunoglobulin G and leaf peroxisomal specific glycolate oxidase immunoglobulin G. The labeling density of citrate synthase was decreased in the microbodies during the greening, whereas that of glycolate oxidase was dramatically increased. Double labeling experiments using different sizes of protein A-gold particles show that both the glyoxysomal and the leaf peroxisomal enzymes coexist in the microbody of the transitional stage indicating that glyoxysomes are directly transformed to leaf peroxisomes during greening.

Journal Article↗
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

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells.

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K(m) for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective ;PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.

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↗
PubMed1986

Light modulation of maize leaf phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH(4))(2)SO(4) fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.

Journal Article↗