Specific Inhibition of Cell Wall-Bound ATPases by Fungal Suppressor from Mycosphaerella pinodes

Activities of phosphatases were found in the fractions whichwere solubilized from cell walls of both pea and cowpea seedlingswith 0.5 M NaCl. These phosphatases hydrolyzed triphosphonucleotidesin the order: UTP=CTP>GTP>ATP; and UTP=GTP>CTP=ATP,respectively. The activities of a pyrophosphatase and a p-nitrophenylphosphatasewere also detected in these fractions. The suppressor in thespore germination fluid of a pea pathogen,Mycosphaerella pinodes,inhibited all of these phosphatase activities in the fractionsolubilized from pea cell walls, but it rather enhanced onlythe activity of the ATPase among those phosphatases from thecowpea cell wall. Hydrolysis of ATP by a cell wall fractionof pea was also markedly inhibited by the suppressor, whilehydrolysis of ATP by similar fractions from cowpea, kidney beanand soybean were rather enhanced by the suppressor, as wellas by the elicitor. Thus, the cell wall-bound ATPases respondedto the suppressor species-specifically. These cell wall-boundATPases seemed to be different from the plasma membrane ATPasesin several respects. The results suggest that plants recognizethe fungal signals not only on their plasma membranes but alsoon their cell walls and, moreover that putative receptors forthe fungal signals might be located close to cell wall-boundATPases or might even be these ATPases themselves. (Received November 16, 1994; Accepted April 20, 1995)     

Properties of Membranes from the Halophyte Suaeda maritima: II. DISTRIBUTION AND PROPERTIES OF ENZYMES IN ISOLATED MEMBRANE FRACTIONS

Membrane fractions from the shoots of Suaeda maritima have beenseparated by sucrose gradient centrifugation. Certain stainingand enzymatic activities were studied with the aim of identifyingspecific membrane types, particularly the plasma membrane. Chloroplastand mitochondrial membranes were largely distributed in thedenser regions of the gradient ( 1.16). A plasma membrane fractionwas not easily identifiable and the problems of separating thesemembranes from plant cells are discussed.     

Subcellular Localization of Enzymes of Carbon and Nitrogen Metabolism in Nodules of Medicago sativa

Alfalfa (Medicago sativa L. cv. Vernal) nodules were separatedinto host plant fractions and fractions of rhizobial originby differential centrifugation and sedimentation equilibriumcentrifugation. Both NAD- and NADP-linked isocitrate dehydrogenase(70%, 90%) and glutamate dehydrogenase activities (90%, 83%)were located primarily (percent total nodule activity) in thefractions of plant origin and their specific activities werehighest in the fractions of plant origin. More than 50% of thenodules' total activity of both glutamine synthetase and NAD-glutamatesynthase and greater than 90% of the total glutamate oxaloacetatetransaminase activity was located in plant fractions. However,the fractions of rhizobial origin had the highest specific activitiesof glutamine synthetase and glutamate synthase. (Received September 5, 1981; Accepted December 7, 1981)     

Properties of Alkaline Phosphatase in Cucumber Roots Induced by Calcium Starvation

Calcium deficiency caused an increase in alkaline phosphataseactivity in cucumber roots [Matsumoto and Yamaya (1981) Plant& Cell Physiol. 22: 1137]. The activities of other hydrolasesincluding acid phosphatase, nucleases and proteases, however,were much less affected by the removal of calcium. Nucleosidedi- and triphosphates and inorganic pyrophosphate were effectivelyhydrolyzed by the induced alkaline phosphatase, whereas nucleosidemonophosphate-hydrolyzing activity was basically equal in theroots grown with either complete medium or a medium lackingcalcium. The alkaline phosphatase in cucumber roots was foundin fractions pelleting at 3,000 x g and in the 100,000 x g supernatant.The calcium-starved roots increased their alkaline phosphataseactivity in both fractions. Four isozyme bands of the alkalinephosphatase in the soluble fraction were separated by polyacrylamidegel electrophoresis. One of the isozyme bands showed a prominentincrease with the calcium deficiency, but not in the presenceof cycloheximide. (Received June 24, 1981; Accepted September 3, 1981)     

The Ultrastructure of the Alkaloidal Vesicles of Papaver somniferum latex

Papaver somniferum latex contains abundant small vesicles. Theirultrastructure was studied in tissue sections from adult plantsand in sections of sequential fractions of centrifuged latex.The vesicles were found to exist in two forms, the first witha smooth but progressively granulated outer membrane and thesecond, probably derived from the first, with adherent ‘cap-like’structures which in the heavier centrifuged fractions possesseda zonally-ordered interior. These vesicle fractions were active in synthesizing morphineand the name ‘alkaloidal vesicle’ is proposed forthem. Papaver somniferum latex also contains an organelle whichwas found to resemble a complex organelle present in the latexof Hevea brasiliensis. Its function is not yet known.     

The Effects of Pectic Enzymes and Phosphatidases on Potato Tuber Tissue

Extracts from rots of potato tubers caused by Erwinia atrosepticaand Corticium praticola were fractionated by precipitation withammonium sulphate and by gel filtration. For the various fractionsof the E. atroseptica extracts there was a close relation betweenthe activity of pectate franj-eliminase and capacity to increasethe permeability of protoplasts as assessed by loss of electrolytes.There was no such relation with phosphatidase acting on lecithin. For certain fractions of C. praticola extracts there was a similarclose relation between increase in permeability and activityof a polygalacturonase but for other fractions with low polygalacturonaseactivity there was a better relation with phosphatidase thoughall fractions that caused increase in permeability did havesome polygalacturonase activity. Phosphatidases which probablyplay no part in the killing of cells in E. atroseptica rotsmay, therefore, have some role in the killing of cells in C.praticola rots though they are likely to be less important thanpectic enzymes. Extracts from E. atroseptica rots caused marked increases inuptake of oxygen by tuber discs. Dialysis decreased and heatingeliminated this increase and had corresponding effects on permeability.However, after fractionation with ammonium sulphate, fractionswith high trans-eliminase activity had little effect on oxygenuptake whereas fractions with low trans-eliminase had littleeffect on permeability and greatly increased oxygen uptake. Similar results were obtained with C. praticola rot extracts.In contrast, nigericin and Triton X-100 both increased permeabilityand caused large increases in oxygen uptake The significance of these results is discussed especially inrelation to the killing of protoplasts by extracts from bothtypes of rot.     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Growth Accelerators and Inhibitors in Lemon Buds Infested by Aceria sheldoni (Ewing) (Acarina: Eriophyidae)

The biological activities of growth accelerators and inhibitorsin lemon buds both infested and uninfested with the citrus budmite, Aceria sheldoni (Ewing), were examined by the Avena test.The growth-promoting activity of the purified ether extractcontaining the auxin was diminished in proportion to the degreeof infestation. Paper chromatography fractions of extracts obtainedfrom infested buds exhibited a strong inhibiting activity onAvena coleoptile elongation in the presence of 0.1 ppm indol-3yl-aceticacid. This inhibiting activity seems to be due to the presenceof a high level of phenolic compounds. The phenol level andits biological activity in both infested and uninfested budswere examined. Infested buds exhibited a higher amount of phenolsthan uninfested ones, the level of the phenols increasing withbud age. The phenols from infested buds inhibited Avena coleoptileelongation to a much greater extent than those from uninfestedbuds. Our results suggest that the presence of the citrus budmite in lemon buds can increase the level of phenols and possiblyother growth depressors in the buds, and that they, in turn,may cause the observed inhibition of growth and developmentof the infested citrus plant.     

Isolation of Avian and Mammalian Pineal Indoles and Antigonadotropic Factors

With mild extraction and separation methods it was possibleto locate and isolate, starting with 100 or 200 g of pinealtissue, different indoles, with mass spectra identical to thoseof synthetic melatonin, 5-HIAA and 5-MIAA. It was also possibleto isolate fractions, which showed, in thin-layer chromatographicstudies, spots with R_f values identical to those of synthetic5-HTL and 5-MTL. A "non-melatonin" antigonadotropin, in an in vivo bioassay,was also isolated from sheep pineals and partially purified. Antigonadotropic and gonadotropic activities, which differ fromthe above described indoles, could be located with the samemethods. Some of these compounds acted on the hypothalamus,while others acted on the anterior hypophysis in vitro.     

STUDIES ON ALGAL CYTOCHROME: I. ENZYMIC ACTIVITIES PERTAINING TO PORPHYRA TENERA CYTOCHROME 553 IN CELL-FREE EXTRACTS

1. Enzymic activities pertaining to Porphyra tenera cytochrome553 were investigated with cell-free extracts of a red alga,Porphyra tenera, and various fractions prepared therefrom.
2. Thealgal extracts were found to be incapable, in the dark,of catalyzingoxidation of reduced cytochrome 553 at a ratesufficient toaccount for the respiratory oxygen-uptake in theintact cell.Oxidation of cytochrome 553 was highly acceleratedon illumination.The former reaction was found to be cyanide-sensitiveand thelatter, cyanide-insensitive. Both activities were foundto belocalized in the particulate fraction of the cell extract.
3. Significantactivities of reduced pyridine nucleotide-cytochromereductasewere discovered in the soluble fraction of the cellextract,the reaction being two or three times faster with TPNHthanwith DPNH as electron donor. There was no reaction withsuccinatein the presence of cytochrome and 2,6–dichlorophenolindophenol.
4. Porphyra tenera cytochrome 553 was shown to be localized inthe plastids of the algal cell.
5. Possible functions of cytochrome553 in the algal metabolismwere discussed.

     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

STUDIES ON COMPONENTS OF RESPIRATORY SYSTEM OF BEGONIA LEAVES

Investigations were made of the properties of diaphorase, cytochromec reductases, cytochrome c oxidase, and other components ofelectron transfer system in various fractions of leaf homogenateof Begonia semperflorens.

1. All the fractions tested showed the existence of cytochromec oxidase, succinic- and reduced diphosphopyridine nucleotide-cytochromec reductases, and diaphorase. Activities of these enzymes werefound to be associated mainly with the particulate fractions.The particulate fractions showed, in particular, a capacityof reducing oxidized cytochrome c with fumarate, malate, -ketoglutarate,ß-hydroxy-butyrate, and citrate.
2. Optimum pH foroxidation of cytochrome c by the particulatefractions was foundto be 5.5, while that for reduction was7.2.
3. The activityof cytochrome c reductase was partially suppressedby malonate.Partial inhibition of cytochrome c oxidase wascaused by azideand cyanide, the inhibitory effects observedbeing strongerwith particulate fractions than with solublefractions.

(Received August 11, 1962; )     

Isolation and Characterization of Golgi Membranes from Suspension-cultured Cells of Rice (Oryza sativa L.)

The isolation of Golgi membranes from suspension-cultured cellsof rice (Oryza sativa L.) was attempted by linear glycerol densitygradient centrifugation. When "burst" membranes in the pelletobtaind after differential centrifugation at 100,000 ? g weresuspended in 20% (w/w) glycerol in 50 mM malate-NaOH (pH 6.0)and loaded onto a linear density gradient of glycerol, whichextended from 30 to 80% (w/w) in 1 mM EDTA in 50 mM glycylglycine-NaOH(pH 7.5), IDPase, a marker enzyme for Golgi membranes, was separatedfrom other membrane markers on the glycerol gradient. In addition,UDPase and GDPase activities overlapped with the peak fractionof IDPase activity. Furthermore, membrane glycoproteins in eachfraction were characterized by lectin-peroxidase staining. ConcanavalinA and lentil lectin, which have the ability to bind to the high-mannosetype of oligosaccharide, bound to glycoproteins distributedin ER membrane fractions, while wheat germ lectin, castor beanlectin, peanut lectin, and Ulex europaeus lectin-I which recognizethe complex type and/or the mucin type of oligosaccharides interactedwith glycoproteins in the Golgi membrane fractions but not withthose in the ER membrane. These results strongly suggest thatthe oligosaccharide structures of glycoproteins in the ER membraneare of the high-mannose type, whereas glycoproteins in the Golgimembrane have modified N-linked and/or O-linked oligosaccharidechains. (Received November 9, 1988; Accepted October 17, 1989)     

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)     

Endogenous Cytokinins in Developing Fruits of Seeded and Seedless Citrus Cultivars

This report describes the isolation and identification of endogenouscytokinins from Citrus ovaries. Cytokinin active fractions wereobtained by extraction with 80% (v/v) ethanol, followed by purificationwith hexane, n-butanol and a polyvinylpyrrolidone and SephadexLH-20 column chromatography. Five fractions with cytokinin activity were found in the organicphase, using the tobacco callus assay. The main active compoundsin these fractions were separated by HPLC, bioassayed and identifiedby GC—MS as ribosyl zeatin, zeatin and isopentenyl adenosine.Hydrolysis of the first fraction with B-glucosidase gave cytokininactive compounds that in paper chromatography had R_F's similarto those of zeatin and ribosyl zeatin. Treating the aqueousphase with alkaline phosphatase produced a cytokinin activecompound that in paper chromatography had the same R_F as isopentenyladenosine indicating that their ribotide was probably the majorphosphorylated cytokinin present in Citrus ovaries. Key words: Citrus, cytokinin fruit set and development     

Characterization of Prolamins from Meadow Grasses: Homology with those of Wheat, Barley and Rye

Prolamin fractions were extracted from seeds of five speciesof temperate meadow grasses. SDS-PAGE showed the presence ofpolypeptidcs with M_rs of 20–30000 in Phieum pratense andDactylis glomerata, and between 30000 and 65 000 in Lolium perenneand in two species of Festuca (F. rubra and F. arundinacea).The fractions had broadly similar amino acid compositions withhigh glutamate+glutamine (29 to 35 mol %) and phenylalaninc(8 to 10 mol %), but there was some variation in the contentsof prolinc (10 to 23 mol %) and several other amino acids includinglysine (0.3 to 1.9 mol %). Automated Edman degradation of twogel filtration fractions from L perenne and one fraction eachfrom the other four species showed single major N.terminal aminoacid sequences. These were homologous with each other, and withthe -typc prolamins of wheat, barley and rye. Key words: Prolamins, meadow grasses, SDS-PAGE     

Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)