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Biomedical subjects

T Akazawa

Publications and source records attributed to T Akazawa.

At least 145 records · Page 8Linked to original sources
PubMed1979

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium.

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.

Journal Article↗
PubMed1979

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase.

Rabbit antiserum against beta-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with beta-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of beta-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-beta-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that beta-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of beta-amylase bound to protein bodies in cereal seeds.

Journal Article↗
PubMed1978

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts.

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [(14)C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP(2)) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [(14)C]leucine into RuP(2) carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.

Journal Article↗
PubMed1978

Photooxidative Damage in Photosynthetic Activities of Chromatium vinosum.

The capacity of photosynthetic CO(2) fixation in the anaerobic purple-sulfur bacterium, Chromatium vinosum is markedly impaired by strong illumination (9 x 10(4) lux) in the presence of 100% O(2). In the absence of HCO(3) (-), decline in activity occurred gradually, with about 40% of the initial activity remaining after a 1-hour incubation. The addition of 50 millimolar HCO(3) (-) to the incubation medium resulted in a measurable delay (about 30 minutes) of the inactivation process. Ribulose-1,5-bisphosphate carboxylase activity and light-dependent O(2) uptake (electron flow) or crude extracts prepared after pretreatment of the bacterial cells with O(2) and light were not affected but the photophosphorylation capacity of either bacterial cells or chromatophores was drastically reduced. The inhibition of photophos-phorylation in the chromatophore preparations was significantly reduced by the addition of either an O(2) (-) scavenger, Tiron, or an (1)O(2) scavenger, alpha-tocopherol. These results suggest that the active O(2) species, O(2) (-) or (1)O(2), might take part in the observed inactivation.The pretreatment of the bacteria with O(2) and light inhibited CO(2) assimilation through the Calvin-Benson cycle, while relatively stimulating the formation of aspartate and glutamate. It also inhibited the conversion of glycolate to glycine, resulting in a sustained extracellular excretion of glycolate. The inactivation of photosynthetic CO(2) fixation by intact cells was enhanced by low temperature, KCN, or methylviologen addition during the pretreatment with O(2) and light. The mechanism(s) of O(2)-dependent photoinactivation of photosynthetic activities in Chromatium are discussed in relation to the possible role of photorespiration as a means of producing CO(2) in the photosynthetic system.

Journal Article↗
PubMed1977

Enzymic formation of glycolate in Chromatium. Role of superoxide radical in a transketolase-type mechanism.

Chromatophores prepared from Chromatium exhibit a light-dependent O2 uptake in the presence of reduced 2,6-dichlorophenolindophenol, the maximum rate observed being 10.8 micronmol (mg of Bchl)-1 h-1 (air-saturated condition). As it was found that the uptake of O2 was markedly inhibited by superoxide dismutase, it is suggested that molecular oxygen is subject to light-dependent monovalent reduction, resulting in the formation of the superoxide anion radical (O2-). By coupling baker's yeast transketolase with illuminated chromatophore preparations, it was demonstrated that [U-14C]-fructose 6-phosphate (6-P) is oxidatively split to produce glycolate, and that the reaction was markedly inhibited by superoxide dismutase and less strongly by catalase. A coupled system containing yeast transketolase and xanthine plus xanthine oxidase showed a similar oxidative formation of glycolate from [U-14C] fructose 6-P. It is thus suggested that photogenerated O2- serves as an oxidant in the transketolase-catalyzed formation of glycolate from the alpha, beta-dihydroxyethyl (C2) thiamine pyrophosphate complex, whereas H2O2 is not an efficient oxidant. The rate of glycolate formation in vitro utilizing O2- does not account for the in vivo rate of glycolate photosynthesis in Chromatium cells exposed to an O2 atmosphere (10 micronmol (mg of Bchl)-1 h-1). However, the enhancement of glycolate formation by the autoxidizable electron acceptor methyl viologen in Chromatium cells in O2, as well as the strong suppression by 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an O2- scavenger, suggest that O2- is involved in the light-dependent formation of glycolate in vivo.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1976

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts.

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.

Journal Article↗
PubMed1975

Further studies on the subunit structure of Chromatium ribulose-1,5-phosphate carboxylase.

Upon alkali exposure Chromatium ribulose-1,5-bisphosphate carboxylase dissociates into constituent subunits, a catalytic oligomer of the larger subunit, A8, and monomeric form of the small subunit B. By sedimentation equilibrium molecular weights of the native enzyme and the catalytic oligomer produced by an alkali treatment were estimated to be 5.11 x 10 5 and 4.29 x 10 5, respectively. To provide information on reversibility of the dissociation by determining whether the enzymically inactive small subunit B of the whole enzyme molecule did indeed exchange with exogenously added subunit B a radioisotopic method was used. After initial alkaline dialysis at pH 9.2 of a mixture of a nonlabeled native enzyme preparation and 14C-labeled subunit B, and the subsequent dialysis at pH 7.0, incorporation of 14C into the recovered native enzyme was determined. Without the alkaline treatment there was no detectable exchange, while after alkaline dialysis for 5 and 10 hr the subunit B exchange was 89 and 82%, respectively. Rabbit antiserum prepared against the catalytic oligomer of the spinach ribulose-1,5-bisphosphate carboxylase, anti-(A) (spinach), inhibited the Chromatium carboxylase and oxygenase activities. This result together with the identical immunoprecipitation lines on an agar plate formed between the antiserum and the Chromatium carboxylase and between the antiserum and the catalytic subunit of the Chromatium enzyme strongly indicated structural near identity of the catalytic subunits of the spinach and Chromatium carboxylase molecules. Results also show that the catalytic site of the Chromatium ribulose-1,5-bisphosphate carboxylase and oxygenase exists in the large polypeptide chain.

Animals↗
PubMed1975

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm.

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.

Journal Article↗
PubMed1975

Photosynthetic activities of spinach leaf protoplasts.

Photosynthetic activities of protoplasts isolated from spinach leaf (Spinacia oleracea L.) were investigated. The protoplasts were stable up to 9 hr, without loss of the original activity of CO(2) fixation (33-75 mumoles CO(2)/mg Chl.hr) and light-dependent O(2) evolution (33-40 mumoles O(2)/mg Chl.hr), when stored in 0.8 m mannitol-0.05 m N-tris (hydroxymethyl)-methylglycine-NaOH buffer, pH 7, at 4 C in dark. The optimum pH of 8.5 for CO(2) fixation reaction carried out in the present experimental condition employed is about the same as that reported for intact spinach chloroplasts. The CO(2) concentration for half-maximal rate of CO(2) fixation by protoplasts. "Km (CO(2))," were determined to be 19.8 mum (pH 7) and 42 mum (pH 8.5) and are similar to those observed for intact spinach chloroplasts. Protoplasts showed postillumination CO(2) fixation. Over-all results indicate that spinach protoplasts are as active as the intact plant leaf tissues in their photosynthetic activities.

Journal Article↗
PubMed1975

Effect of oxygen on photosynthesis by spinach leaf protoplasts.

The photosynthetic CO(2) fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N(2) with one of either air (21% O(2)) or 100% O(2). The inhibitory effect of 100% O(2) was greater than that of air. The mode of inhibition by 100% O(2) and air was competitive with respect to CO(2); Ki(O(2)) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to (14)CO(2) in light after transferring them from N(2) to O(2) atmospheres were examined. There was no detectable (14)CO(2) incorporation into glycolate under anaerobic and O(2) atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts.

Journal Article↗