Inhibition of Membrane-Bound Adenosine Triphosphatase Activity from Nicotiana tabacum L. Leaves with Alkyltrimethylammonium Bromide

Cetyltrimethylammonium bromide (CTAB), a cationic detergent,more effectively inhibited the activity of membrane-bound epidermaladenosine triphosphatase (ATPase) of tobacco (Nicotiana tabacumL. cv. Samsun) leaves than anionic or non-ionic detergents.The inhibition of ATPase activity was highly dependent on thelength of the alkyl chain of alkyltrimethylammonium: CTAB >dodecyltrimethylammonium bromide > n-octyltrimethylammoniumbromide trimethylammonium chloride cetyl bromide, comparedat 10^–4 M. The last three derivatives hardly inhibitedthe activity. CTAB inhibition was equivalent to that due toother cationic detergents, cetylpyridinium bromide and cetylamine, but less than that by gramicidin S and tyrocidine andstronger than that by N,N'-dicyclohexylcarbodiimide and vanadate. These results show that a certain length of the alkyl chain(C_n>12) and the combination of both hydrophobic and chargedgroups of a detergent moiety are indispensable for inhibitingthe membrane-bound epidermal ATPase activity. (Received January 26, 1982; Accepted April 10, 1982)     

Ethylene Production by Cell-Free Extract of the Kudzu Strain of Pseudomonas syringae pv.phaseolicola

A cell-free ethylene-forming system of Pseudomonas syringaepv.phaseolicola (Kudzu strain) was characterized by its psychrophilictrait. Ethylene was most effectively produced from -ketoglutaricacid (-KG) at 0.5 mM followed by glutamate and then istidineat 5 to 10 mM. The presence of FeSO₄ was essential to the cell-freesystem. DTT and histidine greatly stimulated ethylene production;the latter could be substituted to some extent by its analogues.The optimum pH value and temperature for the ethylene-formingreactions were pH 7.0 and 25?C, respectively. Ethylene formationfrom -KG was inhibited in the presence of carbonates or organicacids of the TCA cycle, whereas that from glutamate was inhibitedin the presence of ammonium salts. Ethylene production from-keto--methylthiobutyric acid in the cell-free system was largelydependent on non-enzymical processes in the presence of DTTand FeSO₄. The ethylene-forming reactions were inhibited completelyby 1 mM n-propyl gallate and 1 mM p-chloromercuribenzoic acidand partly by coenzymes such as pyridoxal-1-phosphate, folicacid, and flavin mononucleotide at 5mM. The complete systemfor the highest ethylene production consisted of: 0.5 mM -KG,50 mM HEPES (pH 7.0), 5 mM DTT, 0.5 mM FeSO₄, and 10 mM histidine.The amount of ethylene produced in this system was equivalentto 40 to 50% of that produced by the living cells. (Received October 22, 1986; Accepted January 19, 1987)     

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; )     

A Rapid and Sensitive Method for Determination of Relative Specificity of RuBisCO from Various Species by Anion-Exchange Chromatography

A rapid and sensitive method to determine the relative specificity() of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)using anion-exchange chromatography is described. We employedthe open gas system for the reaction of RuBisCO to get the mostreliable CO₂ and O₂ concentrations in the reaction mixture.3-Phosphoglycerate and 2-phosphoglycolate which were formedin the RuBisCO reaction were completely separated and directlymeasured with anion-exchange chromatography without using radioisotopes.The determination of the value was accomplished in 3 h. The values of RuBisCO enzymes from higher land plants were between90 and 96, and those from bacteria including cyanobacteriumwere close to 45. These values were in agreement with previouslyreported values. The enzyme of the red macroalga Porphyra yezoensisexhibited a value of over 140, as expected from the reportedvalue of the enzyme from the red microalga Porphyridium cruenteum.RuBisCO from the green macroalga Ulva pertusa had a value closeto 70 and similar to that of the enzyme from the green microalgaChlamydomonas reinhardtii. ⁴On leave from Research and Development Center, Unitika Ltd.,23 Kozakura, Uji, Kyoto, 611 Japan. ⁵Present address: Nara Institute of Science and Technology,8916-5 Takayama-Cho, Ikoma, Nara, 630-01 Japan     

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; )     

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)     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis     

The Derivation of the Cell Wall Elasticity Function from the Cell Turgor Potential

An equation is derived expressing average turgor pressure ofa leaf (_p) as a function of relative water content (RWC). Basedon this derivation, the relationships of the bulk elastic modulus(_v) and both RWC and _p, are formulated and discussed. The bulkelastic modulus (_v) becomes zero for _p = 0, that is at the turgorloss point for the leaf. At full water saturation the valueof ev is proportional to the water saturation turgor potential_p(max). The factor relating _P and _v (structure coefficient ,Burstrom, Uhrstr?m and Olausson, 1970) changes only very littlefor values of _p, which are not too close to zero. An exampleis given for the calculation from experimental data of the turgorpressure function, the structure coefficient function, and the_v function. Key words: Cell wall, Turgor pressure, Bulk elastic modulus     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

Purification and properties of soluble cytochrome b-560 from spinach leaves

A b-type cytochrome having an -band at 560 nm was isolated fromspinach leaves (Spinacia oleracea). A method is described forpreparing this cytochrome, cytochrome b-560 (spinach), in apurified state. The cytochrome has, in its reduced state, absorption bands at560 nm (), 530 nm (ß) and 427 nm (); and in the oxidizedstate at 562 nm (), 529 nm (ß) and 417 nm (). Thepyridine ferro-haemochrome prepared from cytochrome b-560 hadan -band at 556.5 nm, indicating the protohaem-nature of theprosthetic group. The cytochrome has an oxidation-reduction potential (E'⁰) of+0.13V at pH 7.0, as measured using the ferri-ferro oxalate system. The cytochrome is rapidly reduced on illumination with red orfar-red light in the presence of spinach chloroplasts and isoxidized at a slower rate in the dark. This photoreduction isinhibited by 1x10^–6 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The molecular weight of the cytochrome is 30,000 asestimated by the dextran gel filtration method. (Received December 3, 1971; )     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Sequential Responses to Water Stress in the Leaf

Water stress was imposed by withholding water at an early vegetativestage from plants of two rice cultivars (IR20 and 63–83)grown in pots. As stress intensified the following sequenceof responses of the leaves was observed: (i) rise in abscisicacid (ABA) content, (ii) closure of stomata, (iii) initiationof leaf rolling. In both cultivars, turgor (_p) declined linearly with total waterpotential () of the leaf. Bulk leaf ABA content increased linearlyas _p declined, and attained twice the control (unstressed) levelfollowing a reduction in _p of about 0.12 MPa. Stomatal conductance exhibited a sigmoidal relationship to _p,declining abruptly when a particular ‘critical’_p was reached (threshold response). The critical potentialsvaried considerably between experiments, but were closely correlatedwith control potentials and with the potentials at which ABAconcentration doubled relative to controls. Leaf rolling was initiated at s near to zero _p. Increases inthe ratio of adaxial to abaxial conductance were associatedwith rolling. Variations in the above responses could be accounted for byvariations in the rate of stress development, which in termsof reduction ranged from 0.38 to 0.86 MPa day^–1. Fastdrying rates resulted in: (a) reduced osmotic adjustment, (b)increased amounts of ABA in the leaf at a given level of or_p, (c) an increase in the ABA concentration present at 50 percent stomatal closure, and (d) initiation of leaf rolling ata higher . Oryza sativa L., rice, water stress, stomata, leaf rolling, abscisic acid     

Water Potential and Mechanical Properties of the Cell Wall of Hypocotyls of Dark-Grown Squash (Cucurbita maxima Duch.) under Water-Stress Conditions

Hypocotyl growth of seedlings of dark-grown squash (Cucurbitamaxima Duch.) was greatly reduced by the addition of 60mM polyethyleneglycol (PEG) to hydroponic solution (water stress). Apoplastic solution (A) and cell sap (C) were separately collectedfrom the hypocotyl segments by a centrifugation method. Theosmotic potentials of A (_A) and C (_c), and (=_c–_A) ofstressed hypocotyls were always lower than those of unstressedhypocotyls. Suction force was measured by immersing the segments into solutionsof different concentrations of mannitol. Suction force was significantlycorrelated with _C (r= –0.99). The mechanical properties of the cell wall of hypocotyl segmentswere measured by stressrelaxation technique. Minimum stressrelaxation time (T_o), relaxation rate (R) and residual stressof unstressed hypocotyls were low during the growth period andincreased when the growth ceased. T_o and R of stressed hypocotylsdecreased one day after the stress treatment, but the residualstress was not decreased by the water stress throughout theexperiment. These results suggest that the suppressed growth of dark-grownsquash hypocotyls under water stress was due neither to thereduction of the osmotic potential difference between innerand outer space of the cell, nor to the decrease in suctionforce, but was partly due to the unchanged mechanical propertiesof the cell wall, as represented by one stress-relaxation parameter,residual stress. (Received February 5, 1988; Accepted September 8, 1988)     

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)     

HORMONAL REGULATION ON THE INDUCTION OF {alpha}-AMYLASE ISOZYMES IN THE EMBRYO-LESS ENDOSPERM OF BARLEY

The -amylase induced by helminthosporol and gibberellic acidin the embryo-less endosperm of barley was separated into thethree fractions, 1, 2 and 3, by DEAE-cellulose column chromatography.A maximum amount of the 2. was induced by gibberellic acid andthat of the as by helminthosporol. After rechromatography, the2 induced by gibberellic acid and the as induced by helminthosporolshowed their respective single bands in an electrophoresis agargel zymogram. On the other hand, the ai induced by helminthosporoland gibberellic acid showed three bands. Dihydrohelminthosporic acid, a derivative of helminthosporol,induced the same isozymes as helminthosporol did. (Received May 8, 1967; )     

Abscisic Acid Accumulation and Water Relations of Four Cultivars of Phaseolus vulgaris L. under Drought

Larqué-Saavedra, A., Rodriguez, M. T., Trejo, C. andNava, T. 1985. Abscisic acid accumulation and water relationsof four cultivars of Phaseolus vulgaris L. under drought.—J.exp. Bot 36: 1787–1792. Plants of four cultivars of Phaseolus vulgaris L. differingin drought resistance were grown in pots under greenhouse conditionsand prior to flowering water was withheld from the pots untilthe mid-day transpiration rate reached values below 1.0 µgH₂O cm^–2 s^–1 (designated the ‘drought’stage). At this point leaves were harvested on 3 or 4 occasionsover 24 h to determine the abscisic acid (ABA) concentration,total water potential (), solute potential (₁) and turgor potential(_p). Results showed that values of , ₁, and _p differed between cultivarswhen they reached the ‘drought’ stage. The stomatalsensitivity to changes in and _p, was as follows: Michoacán12A3 > Negro 150 Cacahuate 72 > Flor de Mayo. These datacorrelated well with the pattern of drought resistance reportedfor the cultivars. ABA accumulation at the ‘drought’ stage differedbetween cultivars at each sampling time, but overall differencesin ABA level between cultivars were not significant. ABA levelsdid not, therefore, correlate with the drought resistance propertiesreported for the cultivars. Results are discussed in relationto and hour of the day when bean samples were taken for ABAanalysis. Key words: Phaseolus vulgaris L., drought resistance, abscisic acid     

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; )     

'Nonparametric inference in multivariate mixtures' * Biometrika (2005), 92, pp. 667 678

The left-hand side of equation (2·8), on p. 671, shouldread {₁ (1 – ₁)}^–1/2 (2₁ – 1) rather than{(1 – ₁)/₁}^1/2 (2₁ – 1). Reflecting this change,the left-hand side of equation (3·1) on the same pageshould be altered to , and the formula at the foot of p. 677 should be modified to {₁ (1– ₁)}^–1/2 (2₁ – 1) + O_p(n^–1/2). No otherformula is affected, and the left-hand side of (2·8)is still increasing in ₁. The numerical results, discussed in4, are influenced in minor ways. In the simulation study, absolutebias is reduced, and variance is either slightly increased orslightly decreased. In the real-data example, using the nonparametricapproach to analysis, mean squared error is further reduced,from 0·0011 to 0·0004. We are grateful to HiroKasahara and Katsumi Shimotsu for pointing out the error.     

Potassium Transport Across the Membranes of Chara: II.42K FLUXES AND THE ELECTRICAL CURRENT AS A FUNCTION OF MEMBRANE VOLTAGE

Smith, J. R. 1987. Potassium transport across the membranesof Chara. II. ⁴²K fluxes and the electrical current as a functionof membrane voltage.—J. exp. Bot. 38: 752–777. The current required to clamp the trans-membrane voltage ofinternodal cells of Chara australis at different levels wasmeasured simultaneously with either the ⁴²K influx or efflux.Examination of the voltage-dependence of the ratio of the electricalcurrent to the unidirectional tracer fluxes yielded no evidenceof any amplification of the electrical driving force on theK⁺ ions. There was thus no evidence for the interaction of K⁺ions with themselves or any other species during their passageacross the membrane. These measurements allow the determinationof , the fraction of the electrical current carried by K⁺ ions.When the external [K⁺] = 10 mol m^–3, the average valueof was 0?85 for V_m > –125 mV and 07?5 for V_m <–150 mV. When the external [K⁺] = 0?1 mol m^–3, was 0?6 for V_m < –80 mV and 0?1 for V_m > –250mV. It was also found that the conductance associated with K⁺transport was inhibited by hyperpolarization. Key words: Potassium, conductance, flux-ratio     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea