Flower-Promoting Effects of Iron and EDDHA in Lemna paucicostata 151

Lemna paucicostata 151 cultured in 1/10 strength M medium containing50 µM FeCl₃ easily flowered in response to short days,although it scarcely flowered under any photoperiod when themedium contained the standard amount of iron (2 µM FeCl₃).The flowering response was accomparied by an increase in theiron content of the plants, which was maximal at pH 5.0. Instandard M medium containing 50 µM FeCl³, this plant didnot flower even though it had a high iron content. Ethylenediamine-di (o-hydroxyphenylacetic acid) (EDDHA) inducedflowering of this strain under continuous light even in theabsence of iron and copper, and its effect was slightly loweredby the presence of iron in the medium. Thus the flower-inducingactivity of EDDHA could not be attributed to the action of ironor copper. EDTA inhibited both the iron uptake and floweringin Fe-rich medium under short-day conditions. (Received May 16, 1986; Accepted July 25, 1986)     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Ethylene, Nitrate and Nitrite Interactions in the Promotion of Dark Germination of Common Purslane Seeds

Ethylene (10 µ1^–1) caused about one-third of highlydark-dormant seeds of common purslane (Portulaca oleracea L.)to germinate in the dark. Attempts were made to increase germinationin the dark with nitrate and ethylene combinations. When applieddirectly to the seeds, KNO₃ did not stimulate germination andKNO₃ plus ethylene did not increase germination above that ofethylene alone. Pre-incubation of seeds in KNO₃ for 4 to 7 dbefore the ethylene applications significantly increased germination.The effects of the KNO₃ pre-incubation were additive at eachof four ethylene concentrations (0.1–100 µ11^–1).Potassium nitrate was effective only when ethylene followedthe KNO₃ pre-incubation period. Potassium nitrite stimulatedabout 25 per cent of the seeds to germinate without a pre-incubationperiod and without ethylene. Also, ethylene plus KNO₂ enhancedgermination above that achieved by either stimulus alone. Silvernitrate did not block the ethylene promotion of germination,but reversed the typical ethylene inhibition of seedling growthfollowing germination. The results support the views that nitrateexerted its effect via conversion to nitrite within the seedand that the rate of nitrate conversion may be a limiting factorin the dark germination of common purslane seeds. Ethylene mayfacilitate nitrite activity by increasing seed sensitivity tothe stimulus. Common purslane, Portulaca oleracea L., ethylene, nitrate, nitrite, germination, dormancy     

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     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)     

Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

We report here the expression in C₂C₁₂ myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 µM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 µM), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ in inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of 35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C₂C₁₂ myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation     

Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl^– secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I_sc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca²⁺-chelating agent BAPTA-AM (50 µM) abolished the increase in I_sc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I_sc was observed. In addition, basolateral treatment with the PGE₂ receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on I_sc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca²⁺-activated KCNN4 K⁺ channel, and the KCNQ1 K⁺ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl^– conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl^– secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl^– secretion requires activation of CFTR and at least two distinct K⁺ channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)     

Tentoxin Inhibits Both Photophosphorylation in Thylakoids and the ATPase Activity of Isolated Coupling Factor F1 from the Cyanobacterium Anacystis nidulans

Tentoxin strongly inhibited the ATPase activity of isolatedcoupling factor 1 (AF₁) from the cyanobacterium Anacystis nidulans,with 50% inhibition occurring at 0.3 µM. When thylakoidsfrom A. nidulans were preincubated with 0.3 µM tentoxinfor 30 min, photophosphorylation was inhibited by 50%. Measurementsof fluorescence from 9-aminoacridine indicated that tentoxininhibited the utilization of the proton gradient by ATP formationin thylakoids. These results indicate that tentoxin is a strongenergy-transfer inhibitor of photophosphorylation in A. nidulans.Tentoxin decreased the level of ATP in intact cells both inthe light and in darkness, its effects being much stronger inthe dark. Tentoxin at 50 µM strongly inhibited the growthof the cells. ³Present address: Corporate Research and Development Laboratory,Tonen Co. 1-3-1 Nishi-tsurugaoka, Ohi-machi, Saitama, 354 Japan ⁴Present address: Technology and Engineering Laboratories, AjinomotoCo., Inc. Suzuki-cho 1, Kawasaki, 210 Japan     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 1986)     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Development of Endopeptidase Activity in Cotyledons of Vigna mungo Seedlings: Effects of Exogenously Applied End-Products and Plant Hormones

The development of endopeptidase activity in cotyledons of Vignamungo seedlings was examined after application of exogenousamino acids, sugars and plant hormones. The endopeptidase activityin the cotyledons fell when germinating seeds were allowed toabsorb a solution of amino acids at high concentrations, andit was postulated that this effect might have been caused inpart by osmotic stress and in part by end-product repression.Protein immunoblotting with an antiserum against SH-EP, themajor cysteine endopeptidase occurring in the cotyledons, showedthat sugars and amino acids at high concentrations also delayedthe post-translational processing of SH-EP intermediates. Endopeptidaseactivity equivalent to nearly twice that in controls was observedwhen GA₃ was applied at 10 to 100 µM to cotyledons thathad been detached from the embryonic axis. In addition, naphthaleneaceticacid at 1 to 100 µM, kinetin at 1 to 10 µM and jasmonicacid at 1 to 10 µM also increased the activity to a limitedextent. Results of pulse-chase experiments suggested that theeffect of GA₁ on the endopeptidase activity in the detachedcotyledons was attributable to suppression of the degradationof the enzyme. Protein immunoblotting revealed the presenceof 34-kOa and 35-kDa intermediates of SH-EP in addition to previouslyreported 36-kDa and 43-kDa intermediates. (Received June 26, 1995; Accepted October 16, 1995)     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)