Mechanisms of the acido- and thermophily of Cyanidium caldarium Geitler VI. Some physicochemical properties of acid and heat stable cytochrome c-553

An acid and heat stable cytochrome of c-type was purified froman acidophilic and thermophilic unicellular alga, Cyanidiumcaldarium. Physicochemical properties and acid and heat stabilitiesof the cytochrome were examined and compared with those of mesophilicalgal cytochromes. ¹ Based on a dissertation submitted to the Tokyo MetropolitanUniversity in partial fulfillment of the requirements for thedegree of Doctor of Science. (Received October 24, 1977; )     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Mechanisms of the acido- and thermo-phily of Cyanidium caldarium Geitler II. Physiological role of the cell wall

1) A partially disintegrated cell preparation of Cyanidium caldarium(i.e. the 100G fraction; cells from which the outer parts ofthe cell wall had been removed) was obtained by differentialcentrifugation of a cell suspension treated with a French press.2) The 100G fraction cells had completely lost the outer partsof their cell walls, but retained their subcellular structureunchanged and had Hill activity that was equally as high asthat of the intact cells. 3) The Hill activity of the 100G fractionshowed an optimum at pH 7.0 and at 35°C. This activity waslost under acid and high temperature conditions (e.g. pH 3,50°C) under which intact cells showed high activity. 4)The Hill activity of the 100G fraction was lost by heat treatment(55°C, 10min), acid-treatment (pH 3.0, 10 min) or pre-illumination(3xl0⁵lux, 30 min) though intact cells were not inactivatedby these treatments. However, no remarkable difference in sensitivitytowards inhibitors was found between the 100G fraction and intactcells. 5) We thus concluded that the cell wall plays an importantrole in the acido- and thermo-phily of Cyanidium cells. (Received August 19, 1974; )     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

Salicylic Acid Levels in Thermogenic and Non-Thermogenic Plants

The natural trigger for heat production in the thermogenic inflorescencesof Sauromatum guttatum Schott (voodoo lily) was recently identifiedas salicylic acid (SA), which induced heat production at levelsas low as 13 ng g f. wt^–1. Since then the levels of SAwere determined in other thermogenic and non-thermogenic plantspecies. In thermogenic inflorescences of five aroid species,and in male cones of at least four thermogenic cycads SA levelsduring heat production exceeded 1 µg g f. wt^–1.SA was not detected in the thermogenic flowers of a water lily,Victoria regia Lindl. (Nymphaeaceae), and Bactris major Jacq.(Palmae). Levels of salicylic acid varied substantially in thefloral parts of seven non-thermogenic species and in the leavesof 27 non-thermogenic species. Amorphophallus campanulatus Blume ex Decne, Arum italicum Mill., Arum dioscoridis Sibth. & Son., Philodendron selloum Koch, Monstera deliciosa Liebm., Encephalartosferox Bertol. f., Encephalartos hildebrandtii A. Br. & Bouché, Encephalartos gratus Prain, Dioon edule Lindl. cv. edule, Dioon edule Lindl. cv angustifolium, Sauromatum guttatum Schott, voodoo lily, Victoria regia Lindl., Bactris major Jack, salicylic acid, thermogenicity, heat production     

Effect of environment pH on storage glucan synthesis in three thermophilic algae

Plectonema nostocorum, a thermophilic cyanophyte which lives under alkaline conditions at pHs approaching 13, forms a storage glucan showing a maximum absorption of its iodine complex almost identical with that of another thermophilic cyanophyte, Oscillatoria princeps, which exists at a more neutral pH, and with that of the acidophilic thermophile, Cyanidium caldarium. Gel electrophoretic patterns of the storage glucan-forming isozymes of Plectonema do not differ essentially from those of Oscillatoria. The a₂ phosphorylase isozyme appears to be primer-independent, and resembles the a₂ isozymes of both Oscillatoria and Cyanidium. The isozymes responsible for forming α-1,6-glucosidic branched linkages in Plectonema are of the b.e. type (able to further branch amylopectin), rather than of the Q type (able to branch amylose only to amylopectin).     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

Synthesis of L(+) Tartaric Acid from 5-Keto-D-gluconic Acid in Pelargonium

5-Keto-D-[1-¹⁴C]gluconic acid, the most effective precursorof L(+)tartaric acid among all labeled compounds which haveever been tested in grapes, was found to be a good precursorof L(+)tartaric acid in a species of Pelargonium. The synthesisof labeled L(+)tartaric acid from D-[1-¹⁴C]glucose in Pelargoniumwas remarkably depressed when a 0.5% solution of D-gluconateor 5-keto-D-gluconate was administered continuously to leavestogether with D-[1-¹⁴C]glucose. Our results provide strong evidence that D-[1-¹⁴C]glucose ismetabolized in Pelargonium to give labeled L(+)tartaric acidvia (probably D-gluconic acid and) 5-keto-D-gluconic acid withoutpassing through L-ascorbic acid. Labeled L-idonic acid was found in young leaves of Pelargoniumwhich had been labeled with L-[U-¹⁴C]ascorbic acid. The synthesisof the labeled L-idonic acid increased when a 0.1% solutionof L-threonate was administered continuously to leaves togetherwith L-[U-¹⁴C]ascorbic acid. Specifically labeled compounds, recognized as the members ofthe synthetic pathway for L(+)tartaric acid from L-ascorbicacid via L-idonic acid in grapes, were administered to youngleaves of Pelargonium. Each compound (2-keto-L-[U-¹⁴C]idonicacid, L-[U-¹⁴C]idonic acid, 5-keto-D-[1-¹⁴C]gluconic acid and5-keto-D-[6-¹⁴C]gluconic acid) was partly metabolized, as ingrapes. The metabolic pathway starting from L-ascorbic acidto L(+)tartaric acid via L-idonic acid, however, did not actuallycontribute to the synthesis of L(+)tartaric acid in Pelargoniumprobably because the activity of each metabolic step was muchlower than that observed in grapes. (Received May 28, 1984; Accepted July 30, 1984)     

Heat Shock Proteins of Cyanobacterium Anacystis nidulans

Cells of Anacystis nidulans grown at 30°C were incubatedwith ¹⁴C-Chlorella protein hydrolysate at the elevated temperatures(30–55°C) and the effect of heat shock treatment onprotein synthesis was studied. Incubation temperatures higherthan 45°C caused a significant decrease in the incorporationof amino acids into proteins. Further, the heat shock treatmentinduced significant changes in the fluorographic profile ofthe newly synthesized proteins. (Received October 25, 1985; Accepted December 4, 1985)     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Heat Shock Induced Change in Protein Ubiquitination in Chlamydomonas

Ubiquitin was purified from pea (Pisum sativum L.) and its antibodywas produced. Western blot analysis showed that the antibodycross-reacted with ubiquitins from a green alga Chlamydomonasreinhardtii, a brown alga Laminaria angustata and a red algaPorphyridium cruentum but not with ubiquitin from a blue-greenalga Synechococcus sp. In Chlamydomonas, the antibody also reactedwith some ubiquitinated proteins including 28- and 31-kDa polypeptides.The isoelectric points of Chlamydomonas ubiquitin and the 28-and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively.The ubiquitinated proteins, including the 28- and 31-kDa polypeptideswere detected after in vitro ATP-dependent ubiquitination ofChlamydomonas cell extract with ^l25I-labeled bovine ubiquitin.Heat treatment of Chlamydomonas cells (>40°C) causeddrastic increase of ubiquitinated proteins with high mol wt(>60kDa), and coordinated redistribution or decrease of otherubiquitinated proteins and free ubiquitin. Quantitative analysisrevealed that the 28- and 31-kDa ubiquitinated proteins showeddifferent responses against heat stress, i.e. the former beingmore sensitive than the latter. (Received July 10, 1988; Accepted October 4, 1988)     

PLA2 stimulation of Na+/H+ antiport and proliferation in rat aortic smooth muscle cells

The proliferative properties and the ability to stimulate theNa⁺/H⁺antiport activity of a secretory phospholipaseA₂ were studied in rat aorticsmooth muscle cells in culture. The requirement of the enzymaticactivity of phospholipase A₂ toelicit mitogenesis was assessed by the use of ammodytin L, aSer⁴⁹ phospholipaseA₂ from the venom ofVipera ammodytes, devoid of hydrolyticactivity. We propose that the proliferative effect is mediated by thesame transduction pathway for both proteins. In particular,1) both secretory phospholipaseA₂ and ammodytin L stimulatedthymidine incorporation in a dose-dependent manner; 2) both proteins affected the cellcycle, as assessed by cell growth and fluorescence-activated cellsorting experiments; 3) bothphospholipase A₂ and ammodytin Lincreased intracellular pH, a permissive factor for cell proliferation,through activation of theNa⁺/H⁺antiport; 4) ammodytin L was able todisplace the ¹²⁵I-labeledphospholipase A₂ from specificbinding sites in a concentration range consistent with that capable ofeliciting a cellular response; and5) the inhibition by heparin wassimilar for both proteins, taking into account the ratio of heparin toprotein. In conclusion, the enzymatic activity of phospholipaseA₂ is not required for thestimulation of mitogenesis. The inhibitory effect of heparin combinedwith its therapeutic potential could help to clarify the role ofphospholipase A₂ in thepathogenesis of several preinflammatory situations.

     

Biosynthesis of alicyclic acids from 14C-labeled glucose and erythrose by isolated cells from Vigna angularis leaves

Mesophyll cells isolated enzymatically from Vigna angularisleaves were fed ¹⁴Cglucose or ¹⁴C-erythrose and the time-courseof ¹⁴C incorporation into shikimic and quinic acids was examined.When ¹⁴C-glucose was fed to the cells, the highest radioactivityin quinic acid was observed after 10 hr of incubation, whilethat in shikimic acid was after 14 hr. In the experiment with¹⁴C-erythrose, the radioactivity in shikimic acid rose strikinglyup to the 3rd hour, but ¹⁴C in quinic acid increased graduallyduring the incubation. The incorporation of ¹⁴C into shikimicacid was enhanced when unlabeled shikimic or quinic acid wassupplied to the cells simultaneously with either ¹⁴C-glucoseor ¹⁴G-erythrose, whereas that into quinic acid was not significantlyincreased by these alicyclic acids. The difference in incorporationrate of ¹⁴C into quinic acid from that into shikimic acid wasmore conspicuous in the isolated mesophyll cells than in theepicotyls of V. angularis seedlings. ¹ Present address: Department of Biology, Faculty of Science,Kumamoto University, Kumamoto 860, Japan. (Received September 22, 1978; )     

Preparation and Characterization of Heat-Stable and Very Active Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium, Synechococcus elongatu

Very active and heat-stable oxygen-evolving photosystem II particleswere isolated from the thermophilic cyanobacterium Synechococcuselongatus by treatment of thylakoid membranes with a non-ionicdetergent, sucrose monolaurate (SML). The particles were analyzedin a comparison with photosystem II particles prepared withß-octylglucoside (OG). The two preparations had similarpolypeptide compositions, which were caracterized by high levelsof polypeptides from phycobilisomes. The ratio of chlorophylla to QA was 45 and there were four Mn atoms and one tightlybound Ca²⁺ ion per QA in the particles prepared with SML. Thepreparations were thermophilic, showing substantial rates ofoxygen evolution at temperatures up to 60°C. The maximumrates attained at 45°C were as high as 6.0 mmoles O₂ mg^–1Chl h^–1. PS II particles prepared with OG were similarlythermostable but were less active in oxygen evolution at alltemperatures examined. Kinetic analysis of flash-induced absorptiontransients revealed that about 22% and 28% of photosystem IIreaction centers were not associated with the functional QBsite in the SML- and OG-particles, respectively. When correctedfor the inactive reaction centers, the maximum rates of oxygenevolution by SML- and OG-particles were 7.7 and 7.0 mmoles O₂mg^–1 Chl h^–1, which correspond to half times of1.9 and 2.1 ms for the first-order electron transfer, respectively.Comparison of these half times with those of the S-state transitionand the release of oxygen indicates that the overall photosystemII electron transport is limited by the reduction of added electronacceptors and not by release of oxygen. ³On leave from National Chemical Laboratory for Industry, Higashi1-1, Tsukuba, Ibaraki 305     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Xylem Transport and Storage of Amino Acids by S.W. Australian Mistletoes and their Hosts

Amino acid composition of xylem (tracheal) sap and ethanolicextracts of shoots of mistletoes (Amyema spp. and Lysiana casuarinae)and their hosts were compared, using material collected in theirnative habitats. Data indicated that certain host xylem soluteswere transferred directly to the parasite xylem, while otherswere either not absorbed or were metabolized prior to transfer.Certain solutes were major constituents of parasite xylem, butundetected or only in trace amount in the host. Shoot aminoacid pools of parasites differed markedly from those of hosts.The mistletoe, Amyema preissii, exhibited differential storageand transport of arginine when parasitizing three differentspecies, but accumulated proline on only two of these hosts.Host- specific amino acids (djenkolic acid in Acacia saligna,and tyramine in Acacia acuminata) were transported and accumulatedin relatively large amounts by the parasite, but were not detectedin other associations. Proline was the major solute of Amyemalinophyllum parasitizing Casuarina obesa, but arginine predominatedin Lysiana casuarinae on the same host. However, when L. csuarinaeparasitized A. linophyllum, in turn parasitic on C. obesa, theLysiana accumulated equal amounts of proline and arginine andmore asparagine than when directly on the Casuarina. Xylem feedingof ¹⁵N-labelled aspartic acid or ¹³N-(amide labelled) asparagineto cut shoots or whole haustoria-bearing plants of the mistletoeA. preissii resulted in 68–73% of the ¹⁵N of aspartateand 24–30% of that of asparagine appearing in ethanol-solubleshoot amino compounds other than the fed solute. ¹⁵N labellingpatterns of detached shoots were not noticeably different fromthat of whole plants suggesting that the haustorium had relativelylittle effect on processing incoming solutes. Alanine, glutamine,and arginine were principal recipients of ¹⁵N from aspartate,alanine and glutamine in the case of fed asparagine. It is estimatedthat 24% of the carbon requirements for dry matter accumulationin Amyema linophyllm were met by intake of xylem sap solutesfrom its host Casuarina obesa. Key words: Amino acids, xylem transport, mistletoes, host: parasite relations, N metabolism