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T Akazawa

Publications and source records attributed to T Akazawa.

At least 127 records · Page 7Linked to original sources
PubMed1983

Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase.

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using beta-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major alpha-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of alpha-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the beta-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.Evidence showing that beta-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.

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PubMed1982

alpha-Amylase biosynthesis: evidence for temporal sequence of NH2-terminal peptide cleavage and protein glycosylation.

During the biosynthesis of secretory proteins, an NH2-terminal peptide, referred to as a signal peptide, is cotranslationally cleaved off after the protein enters the cisternal space of the endoplasmic reticulum. It also has been reported that the core glycosylation reaction of some secretory and viral membrane glycoproteins occurs as a cotranslational event. However, despite a huge amount of work, no decisive answer has been given as to the temporal sequence of proteolytic cleavage of the signal peptide and glycosylation on the polysomes for any secretory or membrane glycoprotein. We show here that proteolytically processed and already glycosylated chains of rice seed alpha-amylase exist on the polysomes; furthermore, our results provide direct evidence that glycosylation is preceded by proteolytic processing during the biosynthesis of the alpha-amylase molecule.

Amylases↗
PubMed1982

alpha-Amylase biosynthesis: signal sequence prevents normal conversion of the unprocessed precursor molecule to the biologically active form.

Recently, we showed that completion of the polypeptide chains on the polysomes isolated from germinating rice seed scutellum in a cell-free translation system can direct the synthesis of (i) unprocessed polypeptide containing the signal sequence (precursor 1), (ii) signal sequence-cleaved but nonglycosylated polypeptide (precursor 2), and (iii) the fully processed and glycosylated form of alpha-amylase molecules. The two precursors as well as the mature form of alpha-amylase can thus be produced in almost the same condition. Here, the binding affinity of the enzyme molecule to the substrate analogue beta-cyclodextrin was used as a probe to compare the conformations of the three distinctly different polypeptide chains produced on the polysomes. It was found that the mature secretory form and the nonglycosylated precursor form (precursor 2) specifically bind to beta-cyclodextrin immobilized on an epoxy-activated Sepharose 6B column but the form that has an attached signal sequence (precursor 1) does not. The results provide evidence that the NH2-terminal signal sequence prevents acquisition of beta-cyclodextrin-binding activity, indicating that, in rice seed alpha-amylase, the signal sequence impairs conversion of the unprocessed polypeptide to the enzymically active configuration.

Amylases↗
PubMed1982

Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts.

Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O(2) per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH(3), which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH(3) reassimilation occurs outside the mitochondria.

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PubMed1982

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation.

The biosynthetic mechanism of alpha-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of alpha-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of alpha-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of alpha-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of alpha-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of alpha-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of alpha-amylase were found to consist of three isozymes. The in vitro translated precursor forms of alpha-amylase consisted of four multiple components. These results indicate that heterogeneity of alpha-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice alpha-amylase isozymes are encoded by multiple genes.

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PubMed1982

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization.

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH(3) are discussed.

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PubMed1982

Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin.

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated gamma' chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

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PubMed1982

Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium.

The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as alpha-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial beta-oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings.

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PubMed1981

Mechanism of glycolate transport in spinach leaf chloroplasts.

The incorporation of (14)CO(2) into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to (14)CO(2) (NaH(14)CO(3), 1 millimolar) in the light was determined as a function of O(2) concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K(m) (O(2)) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic (14)CO(2) fixation under an O(2) atmosphere.Glycolate transport into and out of chloroplasts was studied using the silicon oil centrifugation method. Both uptake and loss of glycolate were found to be rapid, with small temperature dependencies between 0 degrees C and 25 degrees C (Q(10) = 1.1). The reaction rate as a function of the concentration of glycolate up to 30 millimolar was linear in both directions.The effect of external pH on the reaction rate in both directions was also examined. Glycolate penetrates rapidly, even at pH 8, showing a surprisingly high permeation of the glycolate anion. This rate was about 30 micromoles per milligram Chl per hour at 0 degrees C, and the initial concentration of glycolate of 10 millimolar with a pH range of 7 to 8. The observed rate is comparable to the reported value for glycolate synthesis in chloroplasts under photorespiratory conditions. The uptake of glycolate into chloroplasts was accelerated below pH 7, while the rate of excretion was considerably lowered. It is, thus, suggested that undissociated glycolic acid penetrates the chloroplast envelopes more rapidly than does the anion.

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PubMed1981

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM.

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [(35)S]methionine into the alpha-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-alpha-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify alpha-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that alpha-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of alpha-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of alpha-amylase.

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PubMed1980

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical.

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.

Glycolates↗
PubMed1980

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 9. DE NOVO SYNTHESIS OF beta-AMYLASE.

Germinating rice seeds were fed with [(35)S]methionine and the incorporation of (35)S into beta-amylase demonstrated by quantitative immunoprecipitation using rabbit anti-beta-amylase immunoglobulin G fraction. Separation of the antigen-antibody complex by Na-dodecylsulfate gel electrophoresis and subsequent radioautography clearly showed the radioactive labeling of the beta-amylase molecule. The specific radioactivity of beta-amylase derived from scutellum by immunoprecipitation was significantly greater than that of the endosperm. The results strongly indicate that at the onset of germination of rice seeds beta-amylase is synthesized de novo in the scutellum and that in later stages there occurs activation of an inactive, latent form of the enzyme associated with starch granules in the endosperm. In later stages of germination this activated form of the enzyme becomes dominant.

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PubMed1980

Development of Enzymes Involved in Photosynthetic Carbon Assimilation in Greening Seedlings of Maize (Zea mays).

Upon illumination of dark-grown maize seedlings (5 days old) with incandescent light, there occurred a nearly simultaneous increase, after a certain lag period, in the activities of enzymes engaged in the C(4) pathway and the Calvin-Benson cycle. The light-induced biosynthesis of chlorophyll (a and b) precedes the increase in enzyme activities and proceeds without lag phase. A diphasic feature in the elevation of enzyme activities as a function of the intensities of light provided was observed; the increase in enzyme activities was enhanced by light intensities greater than 10(3) ergs per square centimeter per second in comparison with light of lower intensities. Under light intensities greater than 10(3) ergs per square centimeter per second, the simultaneous addition of levulinic acid, which inhibited chlorophyll formation, markedly reduced the increase of enzyme activities. However, neither the diphasic light effect nor the inhibitory effect of levulinic acid was observed with ribulose-1,5-bisphosphate carboxylase. The enzyme activities in the dark-grown maize seedlings were enhanced by a brief irradiation with the red light and the red light effect was reversed by the following far red light treatment. The red light-induced increase in the enzyme activities did not accompany chlorophyll synthesis, and was completely inhibited by cycloheximide, indicating that enzyme synthesis rather than activation might be involved. Light may play a dual role in enzyme induction; one is as an energy source through the photosystems at high intensities and the other is presumably as a signal mediated by phytochrome at low intensities.

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