Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

Light Activation of Pyruvate, Orthophosphate Dikinase in Maize Mesophyll Chloroplasts: A Role for Adenylate Energy Charge

Pyruvate, orthophosphate dikinase (EC 2.7.9.1 [EC] ) was activatedin the light and inactivated following a dark treatment in intactmaize mesophyll chloroplasts. Addition of catalase (100–250units/ml) to the assay medium was necessary to obtain good activationand to keep the enzyme in an active state during illumination.Arsenate and carbonyl cyanide m-chlorophenyl-hydrazone, uncouplersof photophosphorylation, inhibited the activation. Pyruvate,which has been proposed to have a critical role in supportingthe light activation of pyruvate, orthophosphate dikinase, actuallyinhibited the activation. The pyruvate level in the chloroplastsuspension decreased when the enzyme was light-activated. Measurementsof adenylates and pyruvate in the chloroplasts indicated thatthe energy state of the chloroplasts was more important forthe light activation than was the level of pyruvate. ¹Present address: Department of Biochemistry, Faculty of Science,Saitama University, 255, Shimo-Okubo, Urawa, 338 Japan ²Present address: National Institute of Agrobiological Resources,Yatabe, Tsukuba, Ibaraki, 305 Japan (Received May 2, 1989; Accepted October 2, 1989)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Partial purification and some properties of an L-{alpha}-amino-S-caprolactam-hydrolyzing enzyme from Cryptococcus laurentii

A new L-amlnolactam-hydrolyzing enzyme was partially purifiedfrom cells of Cryptococcus laurentii which can grow on L-aminolactamas a carbon and nitrogen source. The enzyme required a bivalentmetal ion, such as Mn²⁺ or Mg²⁺, and its molecular weight wasroughly estimated to be 1.5?10⁵ Some other properties were alsostudied. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University Sumiyoshi-ku, Osaka 558, Japan. (Received June 20, 1977; )     

Distribution of Fallover in the Carboxylase Reaction and Fallover-Inducible Sites among Ribulose 1,5-Bisphosphate Carboxylase/Oxygenases of Photosynthetic Organisms

The biphasic reaction course, fallover, of carboxyla-tion catalysedby ribulose 1,5-bisphosphate carboxylase/ox-ygenase (RuBisCO)has been known as a characteristic of the enzyme from higherland plants. Fallover consists of hysteresis in the reactionseen during the initial several minutes and a very slow suicideinhibition by inhibitors formed from the substrate ribulose-l,5-bisphosphate(RuBP). This study examined the relationship between occurrenceof fallover and non-catalytic RuBP-binding sites, and the putativehysteresis-inducible sites (Lys-21 and Lys-30S of the largesubunit in spinach RuBisCO) amongst RuBisCOs of a wide varietyof photosynthetic organisms. Fallover could be detected by followingthe course of the carboxylase reaction at 1 mM RuBP and thenon-catalytic binding sites by alleviation of fallover at 5mM RuBP. RuBisCO from Euglena gracilis showed the same linearreaction course at both RuBP concentrations, indicating an associationbetween an absence of fallover and an absence of the non-catalyticbinding sites. This was supported by the results of an equilibriumbinding assay for this enzyme with a transition state analogue.Green macroalgae and non-green algae contained the plant-type,fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii,Gona-tozygon monotaenium and Netrium digitus, showed a muchsmaller decrease in activity at 1 mM RuBP than the spinach enzymeand the reaction courses of these enzymes at 5 mM RuBP werealmost linear. RuBisCO of a primitive type Conjugatae, Mesotaeniumcaldariorum, showed the same linear course at both RuBP concentrations.Sequencing of rbcL of these organisms indicated that Lys-305was changed into arginine with Lys-21 conserved. ⁷ On leave from Research and Development Center, Unitika Ltd.,23 Kozakura, Uji, Kyoto, 611 Japan. ⁸ Present address: Department of Applied Biological Chemistry,Faculty of Agriculture, Tohoku University, Tsutsumidori-Ama-miyamachi, Sendai, 981 Japan. ⁹ Present address: National Institute for Basic Biology, Myodaiji,Okazaki, 444 Japan. ¹⁰ Present address: Department of Environmental Biology, TokyoPharmaceutical University, Hachioji, Tokyo, 192-03 Japan.     

Contrasting Sensitivities of Chlorella and Higher Plant Phosphofructokinases to Dilution

A partially purified preparation of phosphofructokinase fromChlorella pyrenoidosa became unstable upon dilution. At pH 7.7and 2°C, enzyme activity remained relatively constant whenthe protein concentration was 12 mg/ml, but at 3 mg/ml almostall activity was lost after 95 min. Greater stability was affordedby lower pH, higher temperature, or the presence of fructose-l,6-P₂,ATP or P₁. Phosphofructokinases from the leaves of spinach,oat and lucerne did not show the instability characteristicsof the Chlorella enzyme. ¹Present address: Department of Biochemistry and Nutrition,University of New England, Armidale, N.S.W. 2351, Australia. (Received December 17, 1980; Accepted November 25, 1980)     

Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

Nitrite-reducing activity of modified cytochrome c-553 from the red alga Porphyra yezoensis Ueda

Crystalline cytochrome c-553 was obtained from Porphyra yezoensisUeda. The cytochrome in areduced form was modified to show anitrite-reducing activity after appropriate treatment with heat,hydrogen peroxide, or photooxidation using methylene blue asthe electron acceptor, but the reducing activity was far lowerthan that of the nitrite reductase isolated from this alga.The modified cytochrome c-553 was autooxidizable and showedan absorption spectrum resembling that of cytochrome c-553 inthe oxidized form except for slight shifts of the absorptionmaximumin the -band region toward shorter wavelengths. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

COMPARATIVE STUDIES ON GROWTH, RESPIRATION, PHOTOSYNTHESIS AND PIGMENT CONTENT IN RHODOPSEUDOMONAS PALUSTRIS

1. Comparative studies were performed on growth, photosyntheticand respiratory activities, and pigment content in Rhodopseudomonaspalustris.
2. The growth of the organism, as influenced by variousculturalconditions such as light, aerobiosis, anaerobiosisand nutritionalfactors was investigated.
3. The respiratoryactivity of the bacterium was found to be higherin dark-growncells than in cells grown in the light. The photosyntheticactivitydid not significantly depend on the growth conditionsof theculture. Cells of younger cultures were found to be moreactivethan those of older cultures, with respect both to respirationand photosynthesis.
4. The pigment content was found to be higherin the light-growncells than in the dark-grown ones. The ratiophotosyntheticactivity/bacteriochlorophyll was significantlyhigher in thelatter than in the former.
5. Light, as well asvarious nutritional factors, was found toexert a marked accelerationon pigment formation, although ithas not yet been possibleto culture cells completely lackingin photosynthetic pigmentsand accordingly in photosyntheticactivity.

¹ Present address: Division of Dermatology and Urology, TokyoMetropolitan Hiroo Hospital, Tokyo. ² Present address: Department of Biology, Saitama University,Urawa. ³ Present address: Department of Biochemistry, School of Medicine,Yokohama University, Yokohama. ⁴ Present address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Tokyo. (Received July 23, 1961; )     

Degradation of Xyloglucan by Wall-Bound Enzymes from Soybean Tissue II: Degradation of the Fragment Heptasaccharide from Xyloglucan and the Characteristic Action Pattern of the {alpha}-D-Xylosidase in the Enzyme System

Hydrolysis of the fragment heptasaccharide (glucose : xylose= 4 : 3) from xyloglucan with an enzyme preparation from soybeancell wall produced a penta- and a trisaccharide. The resultsof fragmentation analysis of these oligosaccharides with Aspergillusoryzae ß-D-glucosidase indicate the following structuresfor the penta- and trisaccharide. The detection of these intermediate products suggested thatdegradation of the heptasaccharide took place by sequentialsplitting of the -D-xylosidic and ß-D-glucosidic linkages.A characteristic action pattern of the a-D-xylosidase in theenzyme preparation was found. ¹Present address: Department of Biology, McGill University,Montreal, Canada. ²Present address: Department of Botany, Iowa State University,Ames, Iowa 50010, U.S.A. (Received August 20, 1982; Accepted December 7, 1982)     

Enzymic synthesis of floridean starch in a red alga, Serraticardia maxima

ADP-glucose: -l,4-glucan -4-glucosyltransferase was obtainedfrom a marine red alga Serraticardia maxima in a form boundwith floridean starch granules. The enzyme catalyzed the transferof glucosyl residue from ADP-glucose, UDP-glucose and GDP-glucoseto floridean starch added as a primer. ADP-glucose was the mostefficient glucosyl donor in the reaction. Maltose was producedby ß-amylolysis of the glucan synthesized by the algalenzyme. The optimum pH for enzyme activity was at 8.4. The enzymewas not obtained in a soluble form from either the chloroplastextract or the whole algal cell extract. Electron micrographsof algal cells revealed that floridean starch granules are localizedexclusively outside chloroplasts. Hence, it appears that mostof the synthetase is present outside chloroplasts. ¹ Contribution from the Shimoda Marine Biological Station, TokyoKyoiku University, No. 202. This work was supported by a Grant-in-Aidfor Cooperative Research from the Ministry of Education, Japan. ² Present address: Department of Biology, Faculty of Science,Science University of Tokyo, Kagurazaka, Tokyo 162, Japan. ³ Present address: Laboratory of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. (Received May 25, 1970; )     

Swelling of Etiolated Oat Protoplasts Induced by cAMP and Red Light

Etiolated oat protoplasts were treated with dibutyryl cAMP tostudy possible function of cAMP in the development by measuringthe protoplast swelling. The mean diameter of protoplasts inthe absence of any chemical treatment was 33.58±1.26(SE) µm, which increased to 36.96±0.86 µmin the presence of 100 µM dibutyryl cAMP. Prostacyclin,a potent activator of adenyl cyclase, also showed a significantswelling effect (diameter 38.01±0.98 µm). Red lightalso elicited the swelling of protoplasts (40.26±0.8µm). ¹Present address: Department of Biology, Pusan National University,Pusan 607, Korea. ²Present address: Department of Horticulture, Cheju NationalUniversity, Cheju 590, Korea. ³Present address: Department of Biological Sciences, Texas TechUniversity, Lubbock, TX 79409, U.S.A. (Received June 29, 1985; Accepted November 18, 1985)     

Amarinin: A New Growth Inhibitor from Luffa amara

A growth inhibitor, amarinin was isolated from the seeds ofLuffa amara. Its formula has been established as 2-deoxy cucurbitacinB by spectrometric analysis coupled with chemical degradation.Amarinin inhibited the growth of the second leaf sheath of riceboth in the absence and presence of GA₃. ¹Present address: Department of Chemistry, Arambag College,P.O. Arambag, Hooghly, West Bengal, India ²Present address: Department of Chemistry, Calcutta University,92, A.P.C. Road, Calcutta 700 009 (Received March 15, 1985; Accepted May 1, 1986)     

Cloning of cDNA of Prunus serotina (R)-(+)-Mandelonitrile Lyase and Identification of a Putative FAD-Binding Site

A full-length cDNA clone encoding the flavoprotein (R)-(+)-mandelonitrilelyase was isolated from a black cherry (Prunus serotina) cDNAexpression library and sequenced. A putative FAD-binding sitewas identified near the N-terminus of this enzyme by comparingits deduced amino acid sequence with those of other FAD- andNAD-binding proteins. ¹Present address: Department of Pharmacology, University ofMedicine & Dentistry of New Jersey, Piscataway, New Jersey08854, U.S.A.     

Activation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by Inorganic Phosphate under Nocturnal Conditions

In vivo activation states of ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO; EC 4.1.1.39 [EC] ) in the dark and light phases were measuredin intact leaves of Phaseolus and radish. The activation statewas high in the dark and comparable to the activation stateunder illumination at saturating light intensity. Then, we examined,using RuBisCO purified from spinach leaves, a mechanism forthe activation of RuBisCO in the dark when the stroma is neutralizedand lossess Mg²⁺ partly. Activation was not obserevd when theenzyme was incubated at air-level CO₂ and 10 mM Mg²⁺ at pH rangingfrom 6.2 to 7.5. However, the activation was highly promotedin this pH range when the activation mixture contained 10 mMinorganic phosphate. The activation state was 50 to 60% betweenpH 7.0 and 7.8 and maximum over pH 8.2 in the presence of 10mM inorganic phosphate. Studies of the initial rate of activationshow that the promotion of activation was through stabilizationof the active form of the enzyme by inorganic phosphate, notby altering the pKa of the activator -amino group of Lys-201.The physiological significance of the activation of RuBisCOby inorganic phosphate in the dark is discussed. ³ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.