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     

Separation and Partial Characterization of Membranes from Prochloron sp.

Two differently colored membrane preparations were separatedfrom the prochlorophyte, Prochloron sp., by mechanical disintegrationof the cells followed by sucrose density gradient centrifugation.An orange-colored preparation, containing zeaxanthin as themajor constituent pigment, seemed to comprise the cytoplasmicmembrane. The other green-colored membrane preparation, containingß-carotene and chlorophyll a and b as major pigmentconstituents, was identified as the thylakoid membrane. Thetwo types of membranes were compared as to their absorptionspectra and buoyant densities. ¹ This work is one of the results of the 8th International Expeditionon Prochloron organized by Dr. R. A. Lewin, University of Californiaat San Diego. ⁵ Present address: Solar Energy Research Group, The Algatron,The Institute of Physical and Chemical Research (RIKEN), Wako-shi,Saitama 351, Japan. ⁶ Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received October 19, 1984; Accepted January 7, 1985)     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

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.     

The Deduced Amino Acid Sequence of Phytochrome from Adiantum Includes Consensus Motifs Present in Phytochrome B from Seed Plants

A cDNA for the phytochrome of the fern Adiantum capillus-venerisL. was cloned and sequenced. The deduced phytochrome is 50{smalltilde}55% identical to phytochromes of seed plants, and 68%identical to Selaginella phytochrome. Regions resemble thosein previously characterized phytochromes from ferns, lower plantsand seed plants. ³Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,Tsukuba-shi, Ibaraki, 305 Japan ⁴Present address: Plant Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,Saitama, 351-01 Japan ⁵Present address: Advanced Research Laboratory, Hitachi, Ltd.,Hatoyama, Saitama, 350-03 Japan     

Biosynthesis of Purine Alkaloids in Camellia Plants

The metabolism of [8-¹⁴C]adenine and [8-¹⁴C]hypoxanthine infour species of Camellia plants was investigated in relationto the synthesis of purine alkaloids, caffeine and theobromine.Young leaves of C. sinensis had the ability to synthesize caffeine,but in C. irrawadiensis, these labelled precursors were incorporatedinto theobromine, not caffeine. No synthesis of purine alkaloidscould be detected in C. japonica and C. sasanqua leaves. Conventional"salvage" and degradation pathways of purines were present inall species of Camellia plants examined. ¹ Present address: Research Center, Mitsubishi Chemical IndustriesLtd., 1000 Kamisida-cho, Midori-ku, Yokohama, 227 Japan. (Received September 29, 1986; Accepted January 22, 1987)     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

Molecular Cloning and Characterization of S-Adenosyl-L-Methionine:Scoulerine-9-O-Methyltransferase from Cultured Cells of Coptis japonica

S-Adenosyl-L-methionine : scoulerine-9-O-methyltransferase (SMT)catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionineto the 9-hydroxyl group of scoulerine during the biosynthesisof berberine. We have isolated functionally active cDNA clones(pCJSMTs) from a cDNA library prepared from cultured cells ofCoptis japonica. The longest cDNA insert (pCJSMT1) had an openreading frame that encoded 351 amino acids, but the calculatedmolecular mass (38,364 Da) of the deduced product was slightlylower than the experimentally determined molecular mass of purifiedSMT. Rapid amplification of the 5' end of the cDNA indicatedthat the full-length cDNA of SMT consisted of 1,458 nucleotidesthat encoded 381 amino acids. When the full-length cDNA wasexpressed in E. coli, the molecular mass of the expressed SMTwas greater than that of native SMT in Coptis cells. This resultsuggests that SMT might be produced in a pre-mature form andprocessed post-translationally. SMT was also found to exhibitsequence homology to other O-methyltransferases from plantsand N-terminal region of the SMT polypeptide appeared to benecessary for enzymatic activity. ¹Present address: High Quality Life Research Laboratories, SumitomoMetal Industries, Ltd., 3-5 Hikaridai, Seika, Sourakugun, Kyoto,619-02 Japan ²Present address: Suntory Research Center, 1-1-1 Wakayamadai,Shimamoto, Mishima-gun, Osaka, 618 Japan ³Present address: Department of Cell Biology, The Scripps ResearchInstitute, La Jolla, CA 92037 U.S.A.     

An Alternative Electron Transfer Pathway Mediated by Chloroplast Envelope

Localization of redox active substance(s) in chloroplast envelopeswas revealed by means of the oxidation of Cyt c by isolatedouter and inner envelope preparations. Irradiated chloroplastsreduced extra-chloroplastic Cyt c probably by an envelope electrontransfer chain. The rate was saturated at a level of about 10µmol (mg Chl)^–1 h^–1 under weak light of 10µEm^–2 s^–1. Cyt c photoreduction was inhibited by DCMUbut not by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)indicating that the plastoquinone site is the junction of photosyntheticelectron transfer chain to envelope redox substance. Completesuppression of the non-cyclic photophosphorylation of thylakoidsby the presence of envelope membranes indicates that there isan alternative electron transfer path-way in envelope membranesthat bypasses over the pH-forming plastoquinone shuttle in thephotosynthetic electron transfer chain. ¹ Present address: Photosynthesis Research Laboratory, Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-0198Japan.     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

The annual cycle of planktonic ciliates in nearshore waters at Signy Island, Antarctica

The abundance and biomass of marine planktonic ciliates in BorgeBay, Signy Island, were determined at monthly intervals betweenApril 1990 and June 1991. At least 24 different ciliate taxawere recorded from samples preserved in Lugol's iodine, includingthe tintinnids Codonellopsis balechi, Cymalocylis convallaria,Laackmaniella naviculaefera and Salpingella sp., and the aloricatetaxa Didinium sp. and Mesodinium rubrum. Ciliate abundance andbiomass exhibited a clear seasonal cycle with high values duringthe austral summer and low values in the austral winter. Abundanceranged from 0.3 10³l^–1 in September to 2.3 10³l^–1in January, while biomass ranged from 0.5 µg C l^–1in October to 12.6 µg C l^–1 in December. Small ciliatesdominated abundance throughout the year, and biomass duringwinter. Larger ciliates contributed most to biomass during summer.Aloricate ciliates were common throughout the year, while tintinnidscontributed substantially to abundance and biomass only duringsummer. Salpingella sp. was the commonest tintinnid, but C.convallariacontributed most to tintinnid biomass. The seasonal patternof ciliate abundance and biomass matched that of chlorophylla concentration and bacterial biomass, suggesting tight trophiccoupling between ciliates and other components of the pelagicmicrobial community. ¹Present address: Scott Polar Research Institute, Universityof Cambridge, Lensfield Road, Cambridge CB2 1ER, UK     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

High Affinity of the 5'-Upstream Region of a Winged Bean Chymotrypsin Inhibitor Gene for Nuclear Matrix

The 5'-upstream region of a winged bean chymotrypsin inhibitorgene (WCI-3b) was found to have a high affinity for nuclearmatrix. The region, named WCI-3b MAR (matrix attachment region),is highly A+T-rich and contains multiple sites interacting withnuclear matrix. A MAR was also found in the corresponding regionof the WCI-x gene, another active gene of the WCI family. SeveralMAR-binding proteins were detected in the wheat nuclear matrix. ⁴Present address: Friedrich Miescher Institute, P.O. Box 2543,CH-4002 Basel, Switzerland. ⁵Present address: Research Institute for Biological Sciences(RIBS), Kayo-cho, Jyobo-gun, Okayama, 716–1241 Japan.     

Effects of Calmodulin Antagonists on the Mitochondrial and Microsomal Ca2$ Uptake in Apple Fruit

In apple fruit, active ATP-dependent microsomal Ca^2$ uptakeand respiration-dependent mitochondrial Ca^2$ uptake were observed. The mitochondrial Ca^2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca^2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent K_m value forCa^2$ in mitochondrial Ca^2$ uptake (K_m=0.35 mM) was similar tothat of mitochondrial Ca^2$-ATPase (K_m=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca^2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa^2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca^2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca^2$-ATPase. The microsomal Ca^2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca^2$ uptake may also be modulated by calmodulin. ¹ Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982)     

CHANGE OF B-TYPE HAEM CONTENT IN RELATION TO PEROXIDASE BIOSYNTHESIS IN INJURED SWEET POTATO ROOTS

The methods of quantitative analysis of b-type haem in plantswere investigated. With an improved method developed was determinedthe haem content in the supernatant, mitochondrial, and microsomalfractions of sweet potato tissue. The activities of peroxidase,catalase, and cytochrome oxidase, as well as the contents ofb-type haem and acid-insoluble nitrogen in the cellular fractionswere determined at different incubation times after cuttingof sweet potato tissue. Peroxidase and catalase increased withtime in each celluler fraction, following a short lag phase.In the mitochondrial fraction, b-type haem, cytochrome oxidase,and acid insoluble nitrogen increased linearly with time. Inthe microsomal and supernatant fraction, b-type haem increasedwith time following a short lag phase. The increase in haemcontent of the supernatant fraction appeared to be associatedwith peroxidase formation. Time course analysis showed that ⁵⁹Fe-incorporation into b-typehaem of the supernatant fraction increased with time and thatincorporation was markedly inhibited by blasticidin S. The incorporationof ⁵⁹Fe into mitochondrial haem did not increase with time andwas not inhibited by blasticidin S. Blasticidin S inhibited⁵⁹Fe-incorporation into microsomal haem. Time course analysisof b-type haem content, ⁵⁹Fe-incorporation into b-type haem,and peroxidase activity suggest that in the injured tissue haemis synthesized from low molecular weight compounds and is incorporatedinto peroxidase as the haem moiety. ¹ This paper constitutes Part 57 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ² Present address: Institute for Plant Virus Research, Chiba.     

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.     

Second positive- and negative phototropism in Vaucheria geminata

Second positive and negative phototropism are found in Vaucheriageminata when the light intensity is very high. The dose-responsecurve of this alga resembles those obtained with Avena coleoptiles,suggesting that the phototropism of both plants occurs througha common initial process. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Sendai 980, Japan. (Received October 13, 1976; )     

Odorant responses of Na+-K+ ATPase activity by preparations from paired turbinals of rat olfactory tissue

Nerve ending particle (NEP) fractions were prepared from homogenatesof rat olfactory epithelium by differential centrifugation.Homogenates were made from each of the four turbinals from theright and left sides. Na⁺-K⁺ ATPase activity and its responsein vitro to seven odorous chemicals was measured for each NEPfraction. Each odorous chemical generated a different patternof response of enzyme activity in the eight fractions. Analysisof the results of enzyme activity perturbation indicated thatthe turbinals are not bilaterally symmetric. ¹ Paper #4590, Mississippii Agricultural Experiment Station,Mississippi State, MS 39762. This work was supported by theOffice of Research and Graduate Studies, Mississippi State Universityand Army Research Office Contract #DAAG 29-80-C-0033.     

ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit

Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca²⁺uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent K_m for Ca²⁺ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa²⁺ transport by protoplast and tonoplast vesicles, respectively.Ca²⁺ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca²⁺ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca²⁺ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent. ¹Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986)