Effect of Monoclonal Antibodies on the in Vitro PFR Dark Reversion of Pea Phytochrome

Extraction as P_FR and immunoaffinity chromatography yieldeda pea phytochrome sample with polypeptide size of 121 kdalton,the same as in a crude extract which was immediately heatedin SDS. A difference spectrum was almost the same as that observedin etiolated pea epicotyls except that A₆₆₆/A₇₃₀ of 1.20 wassignificantly larger. At 10C dark reversion from P_FR occurred,with the decrease in A₇₂₈ being almost equal to the increasein A₆₆₇. The kinetics could be resolved into three first-ordercomponents, the major, slow component accounting for more than90% of the absorbance changes. In the presence of monoclonalanti-pea phytochrome antibodies mAP-1, 3 or 5, which bind awayfrom the chromophore, and mAP-7, which binds near the chromophore,the rate of the major component was reduced at either one orboth wavelengths. None of these antibodies affected the absorptionspectra of phytochrome. In the presence of mAP-9, which is suggestedto bind near the amino-terminus, the absorption at the red-light-inducedphotostationary state was reduced and the rate of dark reversionwas increased, resembling partially degraded phytochrome of114 kdalton, but with no evidence of proteolysis. ¹ Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.     

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)     

The Effect of Temperature on the Energy of Activation of Spinach Ferredoxin-NADP+ Oxidoreductase

A non-linear Arrhenius plot of diaphorase activity of the membrane-boundferredoxin-NADP+ oxidoreductase of thylakoids, showed a breakat 25–30°C, whereas a straight-line was obtained forthe soluble reductase. The E_a values of the thylakoid-boundenzyme was 4–7 and 15–18 kj/mol above and belowthe transition temperature, respectively. In contrast, the E_aobtained for the soluble enzyme was in the range 25–30kj/mol. An Arrhenius plot with similar transition to that ofthylakoids, but with higher E_a values, was obtained when thereductase was isolated as a complex closely associated withthe 17.5 kDa-intrinsic polypeptide. The differences in H* and in S* values below and above the transitiontemperature for thylakoids and the complex were almost the samesuggesting that lipids were not important for the transition. Comparing the H* and S* values for the diaphorase activity ofsoluble and membrane-bound enzyme suggest that the conformationof the latter is more favorable for catalysis. ¹Present address: Depto. de Bioquímica, Fac. de Biologiay C.S.I.C., Univ. de Sevilla, Apdo. 1095, 41080-Sevilla, Spain (Received May 1, 1986; Accepted June 18, 1986)     

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)     

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)     

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; )     

Interactions of seed water content with phytochromeinitiated germination of Rumex crispus (L.) seeds

Light-initiated germination levels of Rumex crispus L. seedswere reduced equally by imbibition in mannitol or polyethyleneglycol 6000 (PEG 6000) solutions of the same w, indicating thatthe effects of each were through w. Reduction of the water contentof the seeds with these osmotica decreased the effectivenessof the far-red absorbing form of phytochrome (P_tr) in causinggermination. However, reduced water content had no effect onthe slopes or saturation points of fluence/response curves whichindicates that has no effect on the number of P_tr receptor sites.The time during which a portion of the seeds were still photoreversibleby far-red light was increased by imbibition in PEG 6000, indicatinga direct effect of w on a reaction involving phytochrome. Noqualitative effect of PEG 6000 on the Onset of secondary dormancywas seen; however, its effect on the relative rate of appearanceof secondary dormancy was equivocal. ¹ Mississippi Agricultural and Forestry Experiment Station cooperating. (Received February 23, 1978; )     

Differences between the Surface Properties of the PR and PFR Forms of Native Pea Phytochrome, and Their Application to a Simplified Procedure for Purification of the Phytochrome

The ability of phytochrome from etiolated pea shoots (Pisumsativum L. cv. Alaska) to bind to various chromatographic adsorbentsand its mobility during non-denaturing electrophoresis wereexamined with phytochrome in either the red light-absorbingform (PR) or the far-red light-absorbing form (P_FR). Preferentialbinding of P_FR to modified hydrophilic polyvinyl resins, suchas butyl Toyopearl, phenyl Toyopearl, Blue Toyopearl (CibacronBlue F3G-A conjugated) and Red Toyopearl (Procion Red HE-3Bconjugated), was observed. A simplified method for purificationof native phytochrome was developed based on the propertiesof P_R and P_FR. P_FR bound preferentially to the hydrophobic adsorbents,to indicate that the surface of P_FR is more hydrophobic thanthat of P_R. A difference in net surface charges between P_R andP_FR was detected by an analysis based on the different mobilitiesof the two forms during non-denaturing polyacrylamide gel electrophoresisin gels prepared with various concentrations of polyacrylamide.The apparent molecular weights of P_R and P_FR, estimated fromthe analysis, were 378 and 419 kilodaltons, respectively. Thedifference suggests that a significant change in molecular shapeoccurs during the photoconversion. The differences in surfaceproperties of P_R and P_FR are discussed. (Received April 20, 1991; Accepted August 26, 1991)     

Spectral response of corn (Zea mays) in root geotropism

The primary roots of corn (Zea mays, Wisconsin hybrid 64A ?22R) show positive geotropism following exposure to light. Thisconfirms the works of other investigators. The curvature responsebegins at about 1 hr following irradiation and reached a plateauat 5 hr. A study of wavelengths 350–760 nm, using energiesof 2.24 ? 10¹⁴ photon cm^–2 and exposure times 60 sec,shows that the most effective light is at 660 nm with lessereffectiveness at 460 and 560 nm. The responses at 660 and 460nm are reversible by a far-red (730 nm) exposure, indicativeof the possible participation of phytochrome. Analyses of freshtips of corn roots with a dual-wavelength difference photometershow the phytochrome content in the root to be about 0.16 (OD) per gram fresh wieght. The requirement of light for thegeotropic growth response of corn roots might be an adaptivephenomenon. The occurrence of photomorphogenic activity in thegreen light should be of concern to those who use green as the"safe" light in "dark" experiments. ¹Work supported by U. S. NASA and U. S. ERDA. (Received September 26, 1977; )     

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; )     

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; )     

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)     

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)     

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; )     

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)     

A comparison of the impacts of various nitrogen sources on acid-base balance in C3 Triticum aestivum L. and C4 Zea mays L. plants

This work aimed to study the impacts of acquisition and assimilationof various nitrogen sources, i.e. NO₃^–, NH₄⁺ or NH₄NO₃,in combination with gaseous NH₃ on plant growth and acid-basebalance in higher plants. Plants of C₃ Triticum aestivum L.and C₄ Zea mays L. grown with shoots in ambient air in hydroponicculture solutions with 2 mol m^–3 of nitrogen source asNO₃^–, NH₄⁺ or NH₄NO₃ for 21 d and 18 d, respectively,had their shoots exposed either to 320 µg m^–3 NH₃or to ambient air for 7 d. Variations in plant growth (leaves,stubble and roots), and OH^– and H⁺ extrusions as wellas the relative increases in nitrogen, carbon and carboxylatewere determined. These data were computed as H⁺/N, H⁺/C, (C-A)/N,and (C-A)/C to analyse influences of different nitrogen sourceson acid-base balance in C₃ Triticum aestivum and C₄ Zea maysplants. Root growth in dry weight gain was significantly reduced bytreatment with 320 µg m^–3 NH₃ in Triticum aestivumand Zea mays growing with different N-forms, whereas leaf growthwas not significantly affected by NH₃. In comparison with C₃Triticum aestivum, non-fumigated C₄ Zea mays had low ratiosof OH^–/N in NO₃^–3-grown plants and of H⁺/N in NH₄⁺- and NH ₄NO₃-grown plants. Utilization of NH₃ from the atmospherereduced both the OH^–N ratios in NO₃^– -grown plantsand the H⁺/N ratio in NH₄⁺ - and NH₄NO₃ -grown plants of bothspecies. Furthermore, Zea mays had higher ratios of (C-A)/Nin NH₄⁺ - and NH₄NO₃-grown plants than Triticum aestivum. Thismeans that C₄ Zea mays had synthesized more organic anion perunit increase in organic N than C₃ Triticum aestivum plants.Within both species, different nitrogen sources altered theratios of (C-A)/N in the order: NH₄NO₃>NH₄⁺>NO₃^–.Fumigation with NH₃ increased organic acid synthesis in NO₃^–- and NH₄⁺ - grown plants of Triticum aestivum, whereas it decreasedorganic acid synthesis in Zea mays plants under the same conditions.Furthermore, these differences in acid-base regulation betweenC₃ Triticum aestivum and C₄ Zea mays plants growing with differentnitrogen sources are discussed. Key words: Acid-base balance, ammonia, ammonium, nitrate, ammonium nitrate, C₃ Triticum aestivum L., C₄ Zea mays L.     

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)     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

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; )