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Biomedical subjects

B B Buchanan

Publications and source records attributed to B B Buchanan.

At least 73 records · Page 4Linked to original sources
PubMed1984

The ferredoxin/thioredoxin system: a key element in the regulatory function of light in photosynthesis.

In addition to its well-established function in supplying the energy for carbon dioxide assimilation, light plays a regulatory role in photosynthesis. The ferredoxin/thioredoxin system is a major mechanism whereby light functions in this capacity. Here, light absorbed by chlorophyll is converted via ferredoxin into a reductant messenger, reduced thioredoxin, that interacts with key target enzymes, thereby changing their catalytic activities. In this way, the green plant achieves maximum efficiency of its photosynthetic (light) and heterotrophic (dark) capabilities.

Adenosine Triphosphate↗
PubMed1983

Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

Carboxy-Lyases↗
PubMed1983

Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin.

Thioredoxins are small redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of important biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Reduced ferredoxin is known to serve as the source of reducing power for the reduction of thioredoxins only in photosynthetic cells that evolve oxygen. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction is considered to be NADPH. We now report evidence that Clostridium pasteurianum, an anaerobic bacterium normally living in the soil unexposed to light, resembles photosynthetic cells in that it uses reduced ferredoxin as the reductant for thioredoxin. Moreover, the transfer of electrons from reduced ferredoxin to thioredoxin is catalyzed by a flavoprotein enzyme that has not been detected in other organisms. Our results reveal the existence of a pathway for the reduction of thioredoxin in which ferredoxin, reduced fermentatively either by molecular hydrogen or by a carbon substrate, provides the reducing power for the flavoprotein enzyme ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin.

Bacterial Proteins↗
PubMed1983

Leaf Cytosolic Fructose-1,6-bisphosphatase : A Potential Target Site in Low Temperature Stress.

Leaf cytosolic fructose-1,6-bisphosphatase (FBPase), partially purified from both spinach (Spinacia oleracea, var Hipack) and peas (Pisum sativum, var Progress No. 9), is reversibly inactivated by exposure to low temperature. Thus, even though assays were conducted at 22 degrees C, samples incubated at 0 to 12 degrees C had greatly reduced activity relative to controls maintained at 22 degrees C. Following incubation at 22 degrees C prior to assay, the inactivated samples regained their initial activity. Chloroplast FBPase, by contrast, was unaffected by low temperature treatment. This feature as well as lack of a response of cytosolic FBPase to thioredoxins f or c(f) and to chloroplast FBPase antibody indicate that the FBPase isozymes of leaves are different proteins.

Journal Article↗
PubMed1983

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana.

Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f(1) and f(2)) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C(3) and C(4) plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C(3), C(4), or CAM photosynthesis.

Journal Article↗
PubMed1983

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana.

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C(3) (spinach) and C(4) (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000-dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.Kalanchoë FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoë ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.Kalanchoë FBPase could be partially activated by Mg(2+) at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca(2+), especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoë SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg(2+) concentration. However, like FBPase, increased Mg(2+) concentrations enhanced the thioredoxin-linked activation of this enzyme.In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoë leaves which required reduced thioredoxin m for activity.These results indicate that Kalanchoë FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C(3) and C(4) plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the functional segregation of glucan synthesis and degradation.

Journal Article↗
PubMed1982

A special fructose bisphosphate functions as a cytoplasmic regulatory metabolite in green leaves.

Fructose 2,6-bisphosphate (Fru-2,6-P(2)), a regulatory metabolite discovered in animal cells and recently reported to occur in etiolated seedlings, was found to be present in the cytoplasmic fraction of leaves of spinach and peas (typical C(3) plants, in which a three-carbon carboxylic acid is a major early photosynthetic product). At concentrations approximating those calculated to occur physiologically, Fru-2,6-P(2) modulated two enzymes of the leaf cytoplasm: (i) Fructose-1,6-bisphosphatase (EC 3.1.3.11), a key enzyme of sugar synthesis, was competitively inhibited by Fru-2,6-P(2), and (ii) pyrophosphate-linked phosphofructokinase (inorganic pyrophosphate-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.90), a cytoplasmic enzyme that now seems important in glycolysis of C(3) plants, was activated by Fru-2,6-P(2). There was no indication of a role for Fru-2,6-P(2) in photosynthesis of either chloroplasts or oxygenic prokaryotes. The results suggest that Fru-2,6-P(2) functions in the regulation of glycolysis and gluconeogenesis (carbohydrate synthesis) in the cytoplasm of leaves of C(3) plants.

Journal Article↗
PubMed1982

Modulation of chloroplast phosphofructokinase by NADPH : a mechanism for linking light to the regulation of glycolysis.

Phosphofructokinase has been partially purified from spinach (Spinacia oleracea) chloroplasts and studied from the standpoint of light/dark regulation. At concentrations reported to occur physiologically, NADPH effected a sharp inhibition of the enzyme by: (a) lowering its affinity (increasing the apparent K(m)) for both of its substrates, ATP and fructose 6-phosphate; and (b) lowering its V(max). Inhibition by NADPH was independent of pH and was observed both at pH 7.9 (pH of chloroplast stroma in the light) and pH 7.0 (stromal pH in the dark). The results are consistent with the conclusion that NADPH provides a mechanism for linking light to the modulation of phosphofructokinase activity and thereby to the regulation of glycolysis in chloroplasts.

Journal Article↗
PubMed1981

Enzyme Regulation in C(4) Photosynthesis : PURIFICATION AND PROPERTIES OF THIOREDOXIN-LINKED NADP-MALATE DEHYDROGENASE FROM CORN LEAVES.

NADP-malate dehydrogenase, a light-modulated enzyme of C(4) photosynthesis, was purified to homogeneity from leaves of corn. The pure enzyme was activated by thioredoxin m that was reduced either photochemically (with ferredoxin and ferredoxin-thioredoxin reductase) or chemically (with dithiothreitol). Unactivated corn leaf NADP-malate dehydrogenase had a molecular weight of 50,000 to 60,000 and was chromophorefree. The enzyme appeared to have a high content of serine and glycine and to contain both S-S and SH groups. Consequently, NADP-malate dehydrogenase seems to be capable of undergoing reversible oxidation/reduction during its photoregulation.

Journal Article↗
PubMed1980

Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION.

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H(2)O as electron donor for the photoreduction of NADP, (b) NADP replaced O(2) as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O(2) at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.The physiological mechanism for the activation of latent polyphenol oxidase remains an unanswered question. Present results suggest that activation could occur through either acidification or the release of free fatty acids.

Journal Article↗