Acetyl-CoA Carboxylase in Higher Plants: Most Plants Other Than Gramineae Have Both the Prokaryotic and the Eukaryotic Forms of This Enzyme

The presence and the absence of a prokaryote type and a eukaryotetype of acetyl-CoA carboxylase (EC 6.4.1.2 [EC] ; ACCase) were examinedin members of 28 plant families by two distinct methods: thedetection of biotinylated subunits of ACCase with a streptavidinprobe, and the detection of the accD gene, which encodes a subunitof the prokaryotic ACCase, by Southern hybridization analysis.The protein extracts of all the plants studied contained a biotinylatedpolypeptide of 220 kDa, which was probably the eukaryotic ACCase.All the plants but those belonging to Gramineae also containeda biotinylated polypeptide of ca. 35 kDa, which is a putativesubunit of the prokaryotic ACCase. In all plants but those inGramineae, the ca. 35 kDa polypeptide was found in the proteinextracts of plastids, while the 220 kDa polypeptide was absentfrom these plastid extracts. The plastid extracts of the plantsin Gramineae contained the 220 kDa polypeptide, as did the homogenatesof the leaves. Southern hybridization analysis demonstratedthat all the plants but those in the Gramineae contained theaccD gene. These findings suggest that most higher plants havethe prokaryotic ACCase in the plastids and the eukaryotic ACCasein the cytosol. Only Gramineae plants might contain the eukaryoticACCases both in the plastids and in the cytosol. The originof the plastid-located eukaryotic ACCase in Gramineae is discussedas the first possible example of substitution of a plastid geneby a nuclear gene for a non-ribosomal component. ⁴Present address: Plant-Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako,351-01 Japan ⁵Present address: Laboratory of Plant Molecular Biology, Schoolof Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya,464-01 Japan     

N-Terminal Amino Acid Sequence and Processing Site of Sweet Potato Cytochrome c Oxidase Subunit II

The N-terminal amino acid sequence of sweet potato cytochromec oxidase subunit II polypeptide was determined. Comparisonsbetween the sequence and amino acid sequences deduced from thenucleotide sequences of other higher plant subunit II genesindicate a post-translational clevage of N-terminal extensionpart. ¹Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan. (Received June 13, 1989; Accepted September 8, 1989)     

Nucleotide Sequence of cDNA Clones Encoding the PS I-H Subunit of Photosystem I in Tobacco

cDNA clones encoding the PS I-H subunit of photosystem I wereisolated from Nicotiana tabacum and Nicotiana sylvestris. Thenucleotide sequences of three clones showed that, in both species,the mature PS I-H protein consists of 95 amino acid residuesand has a calculated molecular mass of 10.3 kDa. ³ Present address: The Institute of Physical and Chemical Research,Tsukuba, 305 Japan.     

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)     

Properties of the Respiratory NAD(P)H Dehydrogenase Isolated from the Cyanobacterium Synechocystis PCC6803

Activity staining with NADPH-nitroblue tetrazolium after native-PAGEof membrane proteins of Synechocystis PCC6803, solubilized with3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),revealed four NAD(P)H dehydrogenase (NDH) activities; an NDHcomplex of the respiratory chain, a ferredoxin NADP⁺ reductase(FNR), a drgA product which oxidized both NADH and NADPH, andan uncharacterized NADH-specific enzyme. The NDH complex waspurified with anion exchange and gel filtration chromatographies.The purified complex had a molecular mass of 376 kDa and wascomposed of 9 subunits. Western analysis showed that the complexcontained the NDH-H subunit, but not NDH-A or B. The enzymereduced ferricyanide much faster than plastoquinone and usedNADPH as its prefered electron donor rather than NADH. The enzymaticactivity was inhibited by diphenyleneiodonium chloride and salicylhydroxamicacid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide,flavon, dicumarol, or antimycin A. These results suggest thatthe purified complex is a hydrophilic subcomplex which containsan NADPH binding site and flavin, and is dissociated from ahydrophobic subcomplex, which contains quinone binding site. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, Hiroshima, 729-0292Japan     

Properties of the Dicyclohexylcarbodiimide-Binding Subunit of Cytochrome c Oxidase from Sweet Potato

Subunit IV of cytochrome c oxidase from sweet potato was boundwith dicyclohexylcarbodiimide and synthesized in isolated mitochondria.Thus, this subunit corresponds to subunit HI of the analogousmammalian and fungal enzymes. Subunit IV was a mixture of twopolypeptides (subunits IVa and IVb) which differed slightlyin structure. ¹Present address: Research Institute of Molecular Genetics,Shimane University, Matsue, 690 Japan. ²Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan.     

Isolation of Chloroplasts from Poteriochromonas malhamensis with the Capacity for Translation

An improved method for the isolation of chloroplasts from Poteriochromonasmalhamensis is described. Poteriochromonas cells were brokenby passage through a nylon mesh with pores of 6µ in diameterat a flow rate of about 5 ml/15 s. After centrifugation thecrude chloroplast fraction was purified by centrifugation ina step gradient of Percoll. The isolated chloroplasts were enclosedby envelope membranes and were still surrounded in part by cytoplasmicresidues. The chloroplasts had the capacity for translation,which was both chloramphenicol-sensitive and cycloheximide-insensitive.The properties of these isolated chloroplasts from Poteriochromonasare discussed in relation to experiments on the transport intothe chloroplasts of nucleus-encoded proteins. ² Present address: Bundesgesundheitsamt, Zulassungsstelle furGentechnologie, Columbiadamm 3, D-1000 Berlin, F.R.G. (Received July 24, 1990; Accepted March 15, 1991)     

Quantification of mRNA in Chloroplasts of Chlamydomonas reinhardii: Equal Distribution of mRNA for a Soluble and a Membrane Polypeptide in Stroma and Thylakoids

The relative contents of the mRNAs were analyzed for the 32kDa herbicide-binding protein and for the large subunit of ribulose-l,5-bisphosphatecarboxylase in the membrane fraction and in the soluble fractionof chloroplasts from Chlamydomonas reinhardii. The presenceof mRNA for the two proteins in both subchloroplast fractionswas demonstrated by in vitro translation of isolated RNA inthe reticulocyte lysate. The relative amounts of the two mRNAswere measured by hybridizations with cloned chloroplast DNAprobes at two stages of the cell cycle. Both mRNAs were distributedin the same ratio between membrane and soluble fractions, about75% of both mRNAs being in the membrane and 25% in the solublefraction. Therefore, in chloroplasts the accumulation of mRNAson thylakoid membranes does not reflect the final localizationof soluble and membrane proteins. ¹Present address: Department of Biology, Ben Gurion University,Beer-Sheva, Israel. (Received April 28, 1987; Accepted September 29, 1987)     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

A Protein Encoded by din1, a Dark-Inducible and Senescence-Associated Gene of Radish, Can Be Imported by Isolated Chloroplasts and Has Sequence Similarity to Sulfide Dehydrogenase and Other Small Stress Proteins

In an attempt to isolate cDNA clones for dark-inducible chloroplastproteins, we screened a cDNA library which was prepared fromradish cotyledons by a two-step method. The source plants weregrown under continuous light for 14 d and kept in darkness for24 h. One of the selected clones, S2D12, corresponded to thedin1 gene which we previously reported as a dark-inducible,senescence-associated gene [Azumi and Watanabe (1991) PlantPhysiol. 95: 577]. A 22 kDa polypeptide was produced from thecDNA in an in vitro expression system in the presence of [³⁵S]methionine.This polypeptide was capable of being imported by isolated chloroplasts,processed to a smaller mature form and localized in the stromalfraction. As the amino acid sequence of the putative matureprotein has no homology to any known chloroplast protein, din1was suggested to be the first gene for a chloroplast proteinwhich is negatively controlled by light. The putative matureprotein has similarity to sulfide dehydrogenase from Wolinellasuccinogenes and other small stress proteins; glpE and pspEfrom Escherichia coli and hsp67B2 from Drosophila melanogaster. ¹ The nucleotide sequence data in this paper has been submittedto EMBL, GenBank and DDBJ Data Libraries under the acces sionnumber AB004242 ² Present address: The Institute of Physical and Chemical Research(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-01 Japan     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

A Factor from Pea Cotyledons That Modifies Auxin-Induced {alpha}-Amylase in vitro

An auxin-induced -amylase (AMY I; EC 3.2.1.1 [EC] ) with a low affinityfor potato starch was purified to homogeneity from detachedcotyledons of Pisum sativum, as judged by the presence of asingle band after non-denaturing PAGE and SDS-PAGE. AMY I wascompared with a previously purified auxin-induced -amylase (AMYII) that had a higher affinity for potato starch. No differencebetween AMY I and AMY II was apparent after SDS-PAGE or isoelectricfocusing (IEF) and rates of degradation of soluble starch wereidentical. However, AMY I was less active than AMY II in thedegradation of starch granules. A factor that converted AMYII to AMY I in vitro was detected in a crude extract of detachedcotyledons. The factor was heat-labile. ¹Present address: Shionogi & Co. Ltd., Fukushima-ku, Osaka,553 Japan     

Isolation and Characterization of the Cytochrome bf Complex from Whole Thylakoids, Grana, and Stroma Lamellae Vesicles from Spinach Chloroplasts

The cytochrome bf complex was isolated from spinach thylakoids,and also from separated grana and stroma lamellae vesicles,by a procedure involving NaBr washing, detergent treatment andcentrifugation in sucrose gradients. The resulting complex fromall three types of membranes, were almost completely devoidof chlorophyll and carotenoids. The complexes have kinase activitytowards histone III-S and contain a 64 kDa protein claimed tobe a kinase. Electrophoretic analyses indicate that the complexesare in dimeric form and composed of six polypeptides with molecularmasses of 34/33, 23, 20, 17, 12 and 4 kDa. The complexes containtwo moles cytochrome b₆ per mole cytochrome f and one mole RieskeFeS. The 17 kDa and 4 kDa polypeptides are the so called subunit4 and 5 respectively. The 12 kDa protein was identified as plastocyaninby immunoblotting. Plastocyanin and the 4 kDa protein were presentin the cytochrome bf complex even after a second repeated sucrosedensity gradient centrifugation. The sucrose gradient sedimentation pattern was different forthe grana and stroma lamellae complexes. The complex from thestroma lamellae arrives at a higher density than the grana complex.Furthermore, the gradient centrifugation diagram of the stromalamellae consists of one main peak while the diagram of thegrana complex shows two peaks. There is significantly more plastocyaninand 4 kDa protein in the bf complex isolated from stroma lamellaethan from grana. In addition there is a 15 kDa protein in thecomplex isolated from the grana vesicles. Immunoblot analysisafter crosslinking indicated that the 4 kDa protein and theplastocyanin are associated in the cytochrome bf complex. Theoxidoreductase activity is higher (about 50%) in the cytochromebf complex from the grana than from the stroma lamellae fraction.We suggest that a difference in composition of the cytochromebf complex between the two membranes might be important in theregulation of cyclic and non cyclic electron flow. ¹Present address: Department of Plant Physiology II, Universityof Warsaw, 00 927 Warsaw, Poland     

Profiiles of Proteins in Guard-cell and Mesophyll Protoplasts from Vicia faba L. Fractionated by Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis

Polypeptides of low molecular weight unique to protoplasts ofVicia guard cells were found by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The polypeptides were 16 kDa (2 species),15 kDa and 12.5 kDa, and were concentrated in membrane-richfractions. The large subunit and holoenzyme of ribulose bisphosphatecarboxylase were identified in guard-cell protoplasts by immunoblotting. ³Present address: Biological Laboratory, College of GeneralEducation, Kyushu University, Ropponmatsu, Fukuoka, 810 Japan. (Received January 25, 1989; Accepted April 24, 1989)     

Chlorophyll-Protein Complexes Associated with Photosystem I Isolated from the Green Alga, Bryopsis maxima

Six chlorophyll (Chl)-protein complexes associated with photosystemI (CPla), and the PS I reaction center complex (CPl) were isolatedfrom the thylakoid membranes of the green alga, Bryopsis maxima,by SDS-polyacrylamide gel electrophoresis. CPla had four polypeptides(22, 24, 25, 26 kDa) in addition to the 67 kDa polypeptide ofCPl. These complexes may thus possibly be a combination of CPland antenna complexes for PS I. Six CPla showed almost the sameoptical properties, with absorption maxima at 650 and 677 nmand contained carotene and a small amount of xanthophylls. TheChl a/b ratios of these CPla were about 2, while that of CPlwas 14. CPla showed a fluorescence emission maximum at 695 nm;its excitation spectrum had peaks at 438, 470 and 540 nm, correspondingto the absorption maxima of Chl a, Chl b, xanthophylls, respectively.An antenna complex free of CPl has been detected in some plantsbut was not found in the present alga. ¹Present address: Department of Botany, The University of Adelaide,Adelaide, S.A. 5001, Australia (Received April 17, 1986; Accepted June 26, 1986)     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

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)     

Sodium Stimulates Regeneration of Phosphoenolpyruvate in Mesophyll Chloroplasts of Amaranthus tricolor

Mesophyll chloroplasts were isolated from leaves of a Na⁺-requiringNAD-malic enzyme type, dicotyledonous C₄ plant, Amaranthus tricolorL. The chloroplasts converted pyruvate to phosphoenolpyruvateunder illumination, and the conversion was stimulated by Na⁺.This observation may explain the requirement for Na⁺ of someC₄ plants. ² Present address: Institute for Life Science Research, NihonNohyaku Co., Ltd., Kawachi-Nagano, Osaka, 586 Japan     

Respiratory Activities, Cytochrome Composition and Cytochrome c Oxidase Subunit II Levels of Mitochondria from Alloplasmic Wheats Having Aegilops Cytoplasms

Respiratory activities, cytochrome composition and cytochromec oxidase (EC 1.9.3.1 [EC] ) subunit II (COXII) levels of isolatedmitochondria in one euplasmic and four alloplasmic lines ofwheat (Triticum aestivum), having cytoplasms of Aegilops columnaris,Ae.crassa (4x),Ae. mutica and Ae. triuncialis, which show variousgrowth abnormalities, were studied. Cytoplasm-specific variationon the respiratory activities has been revealed among the cytoplasmsof five Triticum and Aegilops species. The cyanide-resistantrespiratory pathway does not seem to contribute to the variationin activity. The low level of state 3 and cytochrome oxidaseactivities were evident in lines having Ae. columnaris and Ae.triuncialis cytoplasms, which also show growth inhibition. Thelow cytochrome oxidase activity in both cytoplasms is consistentwith the low amount of cytochrome aa₃ and the severe reductionin COXII levels. The relatively low amount of cytochrome aa₃in Ae. mutica is also consistent with the reduced level of theCOXII. On the other hand, relatively low amounts of cytochromeb (b-557) are found in the Ae. crassa cytoplasmic line. Theseresults suggest that the growth abnormalities found in the alloplasmicwheat lines are related to the variation in respiratory activitiesand cytochrome composition. In particular, growth inhibitionfound in the alloplasmic wheats having Ae. columnaris and Ae.triuncialis may be caused by the severe depression of respiratoryactivity due to the impaired cytochrome oxidase activity oftheir mitochondria. ³Present address: Department of Biochemistry, Dalhousie University,Halifax, NS, Canada B3H 4H7