Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Adaptation of the Euryhaline Charophyte Lamprothamnium papulosum to Brackish and Freshwater: Turgor Pressure and Vacuolar Solute Concentrations During Steady-State Culture and After Hypo-osmotic Treatment

The euryhaline charophyte Lamprothamnium papulosum (Wallr.)J. Gr. was adapted to media with decreasing salinities rangingfrom 550 to 0 mosmol kg^–1. Vegetative plants grown inmedia with osmotic pressures (⁰) in the range of 550 to 130mosmol kg^–1 maintained a constant turgor pressure () at309 + 7 mosmol kg^–1. The ions K+, Na+ and Cl–, werethe predominant solutes in the vacuole. Changes in their concentrationsaccount for the variation in internal osmotic pressure (¹) with,⁰. The divalent ions Mg²⁺, Ca²⁺ and were also present in significant amounts, but their concentrationsdid not alter with changes in, ⁰. In cells subjected to hypo-osmotic shock the regulation of was incomplete. The turgor pressure increased from 302 to 383mosmol kg^–1. The first rapid response to the sudden decreasein ⁰ was a loss of K⁺ and Cl^–. In contrast to the decreasein ionic concentrations an accumulation of sucrose occurredwhich could account for the increase of . The increase in sucroseconcentration started 24 to 48 h after the downshock and reachedits highest value after 3 to 4 weeks. The sucrose concentrationin the vacuole was up to 320 mol m^–3. During this timethe ionic content continued to decrease but did not counterbalancethe sucrose concentration sufficiently to regain the original. High sucrose levels accompanied by an enhanced were also observedduring the period of fructification (sexual reproduction: formationof antheridia and oogonia) in Lamprothamnium kept under conditionsof constant salinity. It is concluded that high sucrose content and elevated arecharacteristic of sexual reproduction in this charophyte. Lamprothamniumis able to tolerate different during various developmentalstages (e.g. vegetative and reproductive phases). Key words: Lamprothamnium papulosum, sucrose, turgor pressure     

Effect of far-red light pulse on induction and production of flowers in Lemna paucicostata 6746 in darkness

A short-day duckweed, Lemna paucicostata 6746, was exposed tocontinuous darkness at 26^?C, and the changes in the floral parameters(3) due to far-red and/or red light pulse given at various timesof the dark period were studied. Parameters a (vegetative growth rate) and (flowering ratio)were respectively decreased and increased with a far-red lightpulse given at the outset of the dark period. The decreaseda and the increased remained almost unchanged until the 7thhour, but returned to their initial levels thereafter. The far-redlight actions on a and were reversed by subsequent exposureto red light. Parameter P₁ (pre-flower induction period) wasextended by 1 day when far-red and/or red pulse was given atabout the 7th hour of the dark period. A far-jed pulse givenat the outset of the dark period only affected parameter P₂(flower induction period). Although the sensitivity of P₂ tored light increased with time, its sensitivity to far-red lightremained constant and at about the 7th hour was equally sensitiveto far-red and red lights. Both red and far-red pulses givenlater than the 7th hour were increasingly ineffective on P₂.The red/far-red reversibility occurred only for the action onP₂ of the far-red pulse applied during the early dark period.Parameter P₄ (flower production period) varied rhythmicallyin length with a far-red puke, the maximum shortening and extensionbeing induced by the pulse given at about the 7th and 19th hours,respectively. The sensitivity of P₄ to red light also changedrhythmically with an inverse phase angle to the rhythmic responseto farred light, and the far-red and red light actions werereversed respectively by subsequent red and far-red lights. These findings suggested that multiple timing devices includingan hourglass-type clock and a circadian clock are involved induckweed flowering. (Received October 25, 1978; )     

Variation in Natural Abundance of 15N among Plant Parts and in 15N/14N Fractionation during N2 Fixation in the Legume-Rhizobia Symbiotic System

Sixteen legumes were grown in N-free media so that N was suppliedentirely by symbiotic N₂ fixation. The plant tissues were analyzedfor natural ¹⁵N abundance (expressed as ¹⁵N per mil relativeto air N₂) with a ratio mass spectrometer. The nodules of desmodium,centro, siratro, soybean and winged bean showed high enrichmentin ¹⁵N (+9), while red clover showed slight enrichment (+2).The nodules of 9 other forage legumes (Townsville stylo, whiteclover, alsike clover, common vetch, Chinese milk vetch, senna,alfalfa, ladino clover, and hairy vetch) showed little enrichmentin ¹⁵N. In all the legumes investigated, particularly in the ureide-transportingplants such as desmodium, centro, siratro, soybean, winged beanand field bean, the ¹⁵N value of the shoots was negative (–3.2).The ¹⁵N value of the shoots in winged bean and field bean variedby about 1 depending on the Rhizobium strains used. The isotopicmass balance of 13 legumes indicated that isotopic fractionationoccurs during N₂ fixation by the legume-rhizobia symbiosis witha preference for ¹⁴N over ¹⁵N, resulting in a ¹⁵N value of –0.2to –2 in the whole plant. The results indicate that ¹⁵N/¹⁴N isotopic discrimination witha preference for the lighter atom may occur in both N₂ fixationand export of fixed N from nodules. ¹Present address: Department of Soils and Fertilizers, NationalAgriculture Research Center, Kannondai, Tsukuba, Ibaraki 305,Japan. (Received October 8, 1985; Accepted April 7, 1986)     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

An {alpha}-Glucan from Suspension-Cultured Rice Cells

An -glucan was isolated from 11-day-old suspension-culturedrice cells by extraction with hot Na-phosphate buffer (pH 6.8).The -glucan had []_D=+234? (C = 0.14, in water) and its averagemolecular weight was estimated to be about 1.4 ? 10⁴, basedon elution characteristics on acalibrated Sepharose CL-6B column.Upon partial acid hydrolysis, the -glucan gave mainly malto-oligosaccharides.The maximum absorption of the iodine complex of the -glucanin the presence of Na₂SO₄ was at 470 nm. The results of hydrolysisby , ß- and iso-amylases and methylation analysisindicated that the isolated -glucan is a highly branched polysaccharidewith an average chain length of 9. The exterior and interiorchain lengths of the -glucan were calculated to be 5 and 3,respectively. (Received July 23, 1986; Accepted February 7, 1987)     

Cl- Fluxes during Osmoregulation in Chara

Measurements of Cl^– influx in cells of Chara corallinashow that control of this flux contributes to the ability ofthis cell to regulate its osmotic pressure. Transcellular osmosiswas used to generate cell fragments with abnormally high ₁,(H-cells), and with abnormally low ₁, (L-cells). Plasmalemmainflux (_oc) was very high in L-cells, and markedly reduced inH-cells. Influx was not affected by the presence of sucrosein the pond water and the consequent reduction in turgor. InH-cells the chloride flux from cytoplasm to vacuole (_cv) wasalso strongly inhibited. It is suggested that control of Cl^–fluxes at both plasmalemma and tonoplast is involved in osmoregulationin these cells. Key words: Chara corallina, osmoregulation, Cl^– flux     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

Effects of l, N6-ethenoadenylates on the regulation of photosynthetic activities by adenylates; A study of the nucleotide binding sites on chloroplast coupling factor 1

Effects of l, N⁶-ethenoadenylates (e-adenylates) were testedon phosphorylation, and electron transport under phosphorylation,arsenylation and quasi-arsenylation (stimulation of electrontransport in the presence of ATP, AMP and arsenate) conditionsin isolated spinach chloroplasts. -ATP as well as ATP partially inhibited ferricyanide reductionthrough binding to the chloroplast coupling factor 1 with anapparent dissociation constant (KD^app) of around 5µM,which was remarkably larger than that for ATP (ca. 2µM).e-ATP at below 500 µM had no effect on phosphorylationbut inhibited quasi-arsenylation in competition with ATP withan apparent inhibition constant (K₁^app) of around 60 µM. -ADP as well as ADP partially inhibited ferricyanide reductionwith a KD^app value close to that for -ATP. -ADP was phosphorylated(the apparent Michaelis constant, K_m^app=80µM) accompanyingstimulation of ferricyanide reduction to the magnitude predicted(P/_e=l). -ADP-arsenylation was also detected by stimulationof ferricyanide reduction. -AMP alone caused little inhibition of ferricyanide reductionas AMP, but competitively depressed the electron transport inhibitionby ADP and ATP with a K₁^app value of around 200 µM. -AMPwas not effective for ADP phosphorylation but inhibited stimulationdue to quasi-arsenylation coupling in competition with AMP K₁^app=150µM Among the possible combinations of adenylates and -adenylatesfor quasiarsenylation, only [ATP+AMP] could couple with theenergy transduction mechanism. Based on the specificity of binding sites to adenylates and-adenylates, an attempt was made to distinguish at least four(two pairs) kinds of binding sites (at least six sites in toto)on the chloroplast coupling factor 1 for photosynthetic energytransduction. When one pair of sites is occupied by the designatedadenylates or -adenylates (allosteric effectors), the couplingfactor is thought to be in a conformation for coupling withthe energy transduction mechanism in the presence of phosphateor arsenate. ¹Presented to the 1st Symposium of Japan Bioenergetics Group,December 19, 1975, Osaka. (Received February 17, 1976; )     

Pumpkin (Cucurbita sp.) seed globulin IV. Terminal sequences of the acidic and basic peptide chains and identification of a pyroglutamyl peptide chain

Pumpkin seed globulin is composed of heterogeneous polypeptidechains, acidic and chains and basic ₁ and ₂ chains (12). This study showed that the basicchains had similar N-terminal sequences, Gly-Leu-Asp-Glu-Thr-Ile-for the ₁ chain and Gly-Leu-Glu-Glu-Thr-Ile- for ₂. On the contrary,the N-terminal sequences of the acidic and chains were dissimilar, Ile-Gln-Gly-Tyr- for the chain and no N-terminal residue for the chain, according to routine terminal analysis. Pyrrolidonylpeptidase digestion of the chain and its thermolysin digestion followed by Edman degradationsrevealed that the N-terminal sequence of the chain was < Glu-Ile-Glu-Gln-Gln-Glu-Pro(Trp,Ser)-. The N-terminal sequences and the C-terminal residuesindicated that the acidic and chains were more heterogeneous than the basic ₁ and ₂ chains.A preliminary study on the degradation of storage globulin isalso presented. (Received November 9, 1979; )     

Induction of sexual agglutinability of {alpha} mating-type cells as the primary action of the peptidyl sex factor from {alpha} mating-type cells in Saccharomyces cerevisiae

The effect of a pure preparation of substance-I_A (S-I_A) whoseamino acid sequence is identical to that of one of the factorpeptides (2), on sexual agglutinability and DNA synthesis wascomparatively studied. The optimum concentration of S-I_A forthe induction of sexual agglutinability of cells of an inducible strain was 1 ng/ml. The inducing action of S-I_A was detectedin 20 min and reached a maximum in 60 min. Only 8.7% inhibitionof DNA synthesis by S-I_A in the same strain was detected in1 hr and 40.4% inhibition in 2 hr at a concentration of 1 µg/ml.These results suggest that the primary action of the peptidyl sex fractor on a mating-type cells is the induction of sexualagglutinabiity. (Received October 25, 1977; )     

Nucleoside Triphosphate(NTP)-Binding Proteins and Endogenous ADP-ribosyl Transferase in Neurospora crassa

Nucleoside triphosphate(NTP)-binding proteins were detectedin the crude extract of mycelia of Neurospora crassa, whichwas treated with 1% Lubrol PX and fractionated by gel filtration.Protein fractions showing the capacity to bind [³⁵S]ATPS or[³⁵S]GTPS were designated as AGN1 to 6. The binding of [³⁵S]ATPSor [³⁵S]GTPS was prevented in the presence of 0.1 mM ATP orGTP except that in fractions AGN1 and 2, the presence of GTPstimulated the binding of [³⁵S] ATPS to ATP(NTP)-binding proteins.ATP or GTP was 1 to 2 orders of magnitude more effective thanCTP or UTP in preventing the binding of [³⁵S]GTPS in AGN1, 2and 5. Among these fractions AGN1, 2, 5 and 6 showed activityto hydrolyze 1 nM [–³²P]ATP or [–³²P]GTP. NTP-bindingproteins bound with [³⁵S]ATPS or [³⁵S]GTPS had lower apparentmolecular weights than the same proteins without bound nucleotide.Proteins bound with [³⁵S]ATPS or [³⁵S]GTPS and those [³²P]ADP-ribosylatedby endogenous ADP-ribosyl transferase in each fraction wereanalyzed by SDS-PAGE. About 20 species of ATP or ATP-GTP-bindingproteins were detected, several of which were ADP-ribosylated.The binding of [³⁵S]ATPS or [³⁵S]GTPS to NTP-binding proteinswas confirmed by the comparison of non-boiled and boiled samplesimmediately before loading to SDS-PAGE. ATP, GTP, CTP or UTPat the concentration of 0.1 mM effectively removed [³³S]ATPSor [³⁵S]GTPS bound to NTP-binding proteins. (Received December 10, 1990; Accepted April 18, 1991)     

Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Uptake of Thallium, a Toxic Heavy-Metal, in the Cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. Leopoliensis) PCC 7942

Uptake of the toxic heavy-metal, thallium, was studied in thecyanobacterium Synechococcus R-2 (PCC 7942) using clinicallyavailable ²⁰¹Tl ⁺. Thallium was found to distribute across theplasmalemma passively, and so the accumulation ratio of theion ([Tl⁺]_i/[Tl⁺]_o) could be used to calculate the apparentmembrane potential (­_i,o) of the cells (^ETI⁺_i,o = ­_i,o).The permeability of the plasmalemma to TI⁺ (^PTI⁺ 1 to 5 nms^–1)is higher than that of K⁺. Valinomycin does not increase thepermeability of TI⁺. Transient changes in the ­_i,o of cells,because of electrogenic transport of ions, could be detectedfrom its effects upon the uptake rate of TI⁺. HCO^–₃ hyperpolarizedSynechococcus cells, whereas NH⁺₄, CH₃NH⁺, and K⁺ led to depolarization.The use of TI⁺ as a reporter of ­_i,o has some inherent limitations.Tl⁺ is toxic at very low concentrations (inhibitory effectsare apparent after about 6 h at concentrations as low as 1 mmolm^–3). The rate of equilibration is slow (t_1/25 to 20 min).Equilibration of TI⁺ takes about 2 h, which limits its valueas a membrane potential probe. Large amounts of TI⁺ bind tothe surface of the cells making the method impracticable formeasuring accumulation ratios of less than about 10 (­_i,o)values smaller than about –60 mV). Cultures continuouslyexposed to Tl⁺ (10 mmol m^–3) eventually become TI⁺ resistantby actively extruding TI⁺ (µTI⁺_i,o= –3±0.2kJ mol^–1) and so thallium cannot be used as a ­_i,oprobe in such cells. (Received October 28, 1997; Accepted August 31, 1998)     

Variable G2 Duration in Root-Meristem Cells of Vicia faba

Scintillation autoradiography was utilized to demonstrate thevariable duration of G₂ in Vicia faba root meristems. The ratioof the longest (max G) to the shortest (min ) duration was 5.4,with min being approximately 2.5 h.     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Lipid Peroxidation Induced by NAD(P)H and NAD+-Dependent Substrates in Soybean Mitochondria

Lipid peroxidation induced by Fe²⁺ADP in soybean mitochondriais stimulated by pyruvate, malate (in the presence of NAD²⁺)and by NAD(P)H. This lipid peroxidation is almost completelyinhibited by EDTA indicating that iron is essential. Also salicylhydroxamicacid, a specific inhibitor of the alternate oxidase, is a stronginhibitor of lipid peroxidation but its effect should be relatedto chelation or to a general antioxidant action. Rotenone doesnot show any effect on the malateNAD²⁺Fe²⁺ADP-induced lipidperoxidation. From the reported data, it is possible to concludethat, in soybean mitochondria, the peroxidation of unsaturatedlipids can be modulated through a balance between the systemssparking lipid peroxidation, like NAD(P)H Fe²⁺ADP, and the systemswhich protect against it, i.e. quinones, maintained at the reducedstate by the substrates. (Received April 20, 1987; Accepted July 21, 1987)