Column Chromatographic Separation of Chlorophyllide {alpha} and Pheophorbide {alpha}

Chlorophyllide a, pheophorbide a, chlorophyll a and pheophytina were separated in a short time by anion-exchange chromatographywith a short column of DEAE-Sepharose CL-6B. (Received February 16, 1984; Accepted April 13, 1984)     

Purification and Characterization of Rice Embryo BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase)

Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984)     

Changes in Triterpenoid Content during the Growth Cycle of Cultured Plant Cells

Similar changes in the pentacyclic triterpenoid contents wereobserved during the growth cycle of Datura innoxia, Luffa cylindricaand Lycopersicon esculentum seedling callus cells in batch culture.Triterpenoid contents decreased for several days after callusinoculation, then increased rapidly during the mid and lateexponential phases of growth. (Received May 28, 1984; Accepted September 13, 1984)     

Endogenous Abscisic Acid in torosa-2 Mutant Tomato Plant

Analyses of abscisic acid, phaseic acid and dihydrophaseic acidby high performance liquid chromatography were carried out onto-2 tomato mutant plant, which has strong apical dominanceexpression. Significant differences in comparison to the normalplant were found only at 20 days after sowing. Of particularinterest for the to-2 phenotype trait was the high quantitiesof these substances in the roots of this mutant. (Received April 20, 1984; Accepted November 21, 1984)     

A Possible Mode of Calcium Involvement in Dark- and Light-Induced Leaflet Movements in Cassia fasciculata Michx.

The calcium chelator ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited thedark-induced (scotonastic) and light-induced (photonastic) leafletmovements of Cassia fasciculata, while the calcium-ionophoreA 23187 increased the rates of both movements. At concentrationas low as 10^-6M, the inhibitors of calmodulin-mediated reactionstrifluoperazine and calmidazolium (R 24571) also promoted bothmovements. The present results support the hypothesis that calciumis involved in these movements through a calmodulin-dependentreaction. (Received July 7, 1984; Accepted September 14, 1984)     

Purification of C-Type Cytochromes from a Thermophilic Blue-Green Alga, Synechococcus vulcanus, by Hydrophobic Column Chromatography using Toyopearls

Two C-type cytochromes, c-550 and c-553, were extracted by animproved procedure from a thermophilic blue-green alga, Synechococcusvulcanus, and effectively purified by a two-step hydrophobicchromatography. The first step was performed with a ToyopearlHW-65C :ammonium sulfate-66 column and the second with a butyl-Toyopearl650 column. This work is the first to apply butyl-toyopearl650 to protein purification. (Received July 2, 1984; Accepted September 13, 1984)     

Effects of Moniliformin on Mitosis in Maize (Zea mays L.)

Maize (Zea mays L. ‘Norfolk White’) roots were treatedwith solutions of moniliformin (a metabolite of Fusarium moniliformeSheldon) at 0.0001 M and 0.001 M for 8, 24, and 48 h. Only aslight inhibition of division was noted in root tips treatedwith the lower concentration. The higher concentration causeda disruption of the spindle apparatus and consequent C-mitosis,and metaphase accumulation. (Received February 14, 1984; Accepted May 18, 1984)     

The Effect of Infection by Erysiphe graminis f.sp. hordei on Protein Synthesis in Vivo in Leaves of Barley

The effect of infection by Erysiphe graminis f.sp. hordei onprotein synthesis in a susceptible cultivar of Hordeum vulgarehas been studied using in vivo labelling. Results have indicateda decline in protein synthesis in the chloroplasts at 1, 3 and5 days after inoculation and in the cytoplasm at 3 and 5 daysafter inoculation. The most extensive effect of the pathogenwas on the synthesis of a 32 Kd protein synthesised in the chloroplasts. (Received May 16, 1984; Accepted July 10, 1984)     

Factor(s) Protecting Pyruvate Orthophosphate Dikinase of Panicum maximum against Cold-Inactivation

An apparent seasonal change in the cold-lability of pyruvateorthophosphate dikinase was observed in Panicum maximum thatsuggests a climatic change in the amount of the factor(s) whichprotects the enzyme against cold-inactivation. This factor(s)was partially characterized as a large molecule(s) with a heat-labilemoiety essential for its function. (Received May 23, 1984; Accepted July 25, 1984)     

Promotion of Rooting in Azukia Cuttings by Possible Glycoproteins Extracted from Lentinus edodes Culture

Macromolecules purified from Lentinus edodes mycelia cultureenhanced adventitious root formation in Azukia epicotyl cuttings.Partial purification by sequential column chromatographies gavematerial composed of 71% polysaccharides and 29% proteins. Thesugar moiety consisted of mainly xylose, glucose and arabinose,the sum of their contents being more than 76% of the total carbohydrates.The protein moiety consisted of mainly glycine and serine, whichaccounted for more than 40% of the total amino acid residues. (Received June 9, 1984; Accepted November 12, 1984)     

Action of Thiol Proteinase on Nitrate Reductase in Leaves of Hordeum distichum L.

Nitrate reductase (NR) from the leaves of Hordeum distichumwas very susceptible to inactivation by barley leaf thiol proteinase,trypsin, and papain. A cytochrome c reductase species with amolecular weight of about 40,000 was derived from the NR complexby the proteolytic actions. The barley proteinase seemed toattack the Mo⁺-containing component of NR, just like trypsinand papain. It showed a preference for the alanine and tryptophanesters among the carbobenzoxyamino acid-nitrophenylesters tested. In vivo NR activity in the presence of leupeptin was fairlyhigher than that in its absence. Leupeptin also protected NRfrom its cleavage to small cytochrome c reductase species, suggestingthat the barley proteinase may play a role in the in vivo changein NR activity. (Received May 21, 1984; Accepted September 10, 1984)     

Inhibition of Porphobilinogen Synthase Activity by 4,5-Dioxovalerate

The effect of 4,5-dioxovaleric acid on the activity of porphobilinogen(PBG) synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) of the porphyrin synthetic pathway was studiedwith the enzyme purified from Chlorella regularis. 4,5-Dioxovalericacid, a metabolite of 5-aminolevulinic acid, competitively inhibitedPBG synthase with a K_i value of 1.4 mM. The concentration forthe half inhibition of 4,5-dioxovaleric acid (7 mM) was slightlylower than that for the known competitive inhibitor, levulinicacid (12 mM). (Received October 8, 1984; Accepted December 13, 1984)     

Location of the Electron Accepting Site of a b-Type Cytochrome Oxidase in Purified Chromatophore Membranes and Spheroplast Membrane Vesicles from Photosynthetically Grown Rhodopseudomonas sphaeroides

Purified chromatophore membranes and spheroplast membrane vesicleswere prepared from Rhodopseudomonas sphaeroides by the methodof Michels and Konings (1978). The cytochrome c oxidase activityof the spheroplast membrane vesicles was about 15-fold higheron a bacteriochlorophyll basis than that of the chromatophoremembranes. The cytochrome c oxidase activity of the chromatophoremembranes was greatly stimulated (about 10-fold) by an additionof Triton X-100 (0.1%), but there was less stimulated (about1.5-fold) in the case of spheroplasts. The pH optimum of theoxidase activities of the spheroplast membrane vesicles andof chromatophore membranes treated with 0.1% Triton X-100, andthe salt dependencies of the activity of both preparations werethe same. These results show that the membrane-bound b-type cytochromeoxidase of this bacterium accepts electrons from ferrocytochromec on the periplasmic side of the membrane (on the outer surfaceof the spheroplast membrane vesicles). These results also areconsistent with the fact that cytochrome c₂ is located in theperiplasmic space in this bacterium. (Received January 24, 1984; Accepted April 24, 1984)     

Inhibition of Cytokinin-Induced Adventitious Bud Initiation in Torenia Stem Segments by Inhibitors of Serine Proteases

Application of di-isopropyl fluorophosphate (DFP), a highlysensitive inhibitor for serine enzymes, strongly inhibited cytokinin-inducedadventitious bud initiation in Torenia stem segments culturedin vitro. The inhibitory effect was not evident when DFP wasapplied after 3 days of culture. Amount of DFP-binding proteinsremarkably increased in superficial tissues of explants culturedfor 3 and 4 days on a medium containing benzyladenine. At least14 kinds of DFP-binding polypeptides were detected by SDS-polyacrylamidegel electrophoresis and fluorography. DFP-binding to some ofthese polypeptides was inhibited by a prior treatment with phenylmethylsulfonylfluoride and N-p-tosyl-L-lysine chloromethyl ketone. From theseresults, it was suggested that some serine proteases might berelated with biochemical events occurring during the initialstage of adventitious bud differentiation in Torenia stem segments. (Received May 8, 1984; Accepted July 5, 1984)     

Influence of Zeatin on Flowering in Root Forming Cuttings of Anagallis arvensis L.

Applied zeatin affected flowering in cuttings of Anagallis arvensis(long-day plant). According to the zeatin concentration andthe root formation stage, zeatin inhibited flowering at differentdegrees or slightly promoted it. Thus, the root primordium settingseemed to be correlated with this effect of zeatin. (Received February 14, 1984; Accepted May 18, 1984)     

Molecular Sizes of the Catalytic Units of Oxygen Evolution and the Reaction Center in Photosystem II of Spinach Thylakoids

Target analysis of the PS II reaction in spinach thylakoidsshowed that the respective molecular masses of the catalyticunits for oxygen evolution and the reaction center are about120 kDa and 250 kDa based on a kinetic separation of the tworeaction rates. The size of the oxygen-evolving enzyme agreedwith that determined for the PS II preparation from a thermophiliccyanobacterium by the same means [Nugent and Atkinson (1984)FEBS Lett. 170: 89]. Single hit-inactivation of oxygen evolutionand the PS II reaction center units indicates that each functionis driven by a structurally assembled unit. (Received August 6, 1984; Accepted December 17, 1984)     

Purification and Some Properties of Soluble Cytochromes, Cytochrome c-551 and Cytochrome c-550, from a Facultative Methylotroph, Protaminobacter ruber strain NR-1

Two soluble cytochromes of the C-type, cytochrome c-551 andcytochrome c-550, were purified from the bacteriochlorophyll-containingcells of a facultative methylotroph, Protaminobacter ruber StrainNR-1, by ion-exchange chromatography and gel-filtration. Cytochrome c-551 had absorption maxima at 551, 522 and 416 nmin the reduced form, and at 525, 410 and 273 nm in the oxidizedform. This cytochrome was a slightly basic protein with an isoelectricpoint of 8.4. It had a mid-point redox potential of 272 mV atpH 7.0. The molecular weight of this protein was 13,500 and13,700 by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) and gel-filtration, respectively. Cytochrome c-550 had absorption maxima at 550, 522 and 415 nmin the reduced form, and at 527, 409 and 278 nm in the oxidizedform. This cytochrome was acidic, having an isoelectric pointof 4.3. It had a mid-point redox potential of 227 mV at pH 7.0.Its molecular weight was 19,500 and 22,000 by SDS-PAGE and gel-filtration,respectively. (Received August 4, 1984; Accepted October 22, 1984)     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Subchloroplast Fractions from the Brown Alga Fucus serratus: Phosphatidylglycerol Contents

Five pigment-protein complexes were isolated from digitonin-solubilizedthylakoids of Fucus serratus using sucrose density gradientcentrifugation. Two heavy fractions corresponded to PS I andPS II antennae with their active reaction-centers. A light fractionwas identified as a light-harvesting antenna, while the twoother fractions were not very well defined. The light-harvestingfractions contained the major part of the C_16:1-trans-phosphatidylglycerol.The results are discussed and compared with those from Chl a/bplants taking into account the basic organization of brown algachloroplasts. (Received May 15, 1984; Accepted October 25, 1984)     

Phytochrome Control of Its Own Synthesis in Pisum sativum

An analysis of phytochrome synthesis in Pisum seedlings by measuringthe activity of polysomal polyadenylated RNA (poly-A⁺-RNA) codingfor phytochrome apoprotein showed phytochrome control of itsown synthesis; brief red-light irradiation of pea seedlingsinhibited the activity of the RNA, and the red-light effectwas red/far-red reversible. ⁴ Permanent address: Biology Department, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 13, 1984; Accepted September 17, 1984)