COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

EFFECT OF SOME SULFHYDRYL REAGENTS ON LIGHT-INDUCED CO2-FIXATION IN CHLORELLA

1. Using intact cells of Chlorella ellipsoidea, investigationswere made on the effects of some SH-reagents (IAA, CMB and arsenite)upon various reactions pertaining to the primary photogenicagent (designated by R) formed in the mechanism of photosynthesis.
2. The pre-illumination experiments using ¹⁴CO₂ as a tracer haveled us to the inference that (i) the process of photochemicalformation of R is inhibited by IAA, that (ii) the spontaneousdecay of R in the dark is markedly accelerated by CMB and arsenite,but not at all affected by IAA, and that (iii) the participationof R in the cyclic path of carbon leading to the fixation ofCO₂ is not affected by IAA and CMB.
3. Based on the assumptionthat R is a reducing agent, it was discussedthat the fact mentionedunder (iii) is incompatible with theidea that the reductionof PGA to triose phosphate be the soleor rate-determining reductivestep in the cyclic path of carbonin photosynthesis.
4. The possibilitythat R may have a functional SH-group (s) wasinvoked to accountfor the observation that the decay of R inthe dark was markedlyaccelerated by CMB and arsenite.

(Received February 11, 1960; )     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

NITRATE REDUCTASE IN PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM RUBRUM. ADAPTIVE FORMATION OF NITRATE REDUCTASE

1. A method was discovered for adapting the cells of Rhodospirillumrubrum to grow on a nitrate medium, a capacity initially lackingin the organism. The adapted cells were able to grow with nitrateas the sole source of nitrogen. The growth responses of theadapted cells towards various nitrogenous sources were investigatedunder various conditions of incubation (aero- and anaerobiosis,light and dark).
2. The adapted cells were found to have simultaneouslyacquiredthe capacity for reducing nitrite and hydroxylamineas wellas nitrate. The path of nitrogen in the adapted cellswas assumedto be as follows: NO₃^– NO₂^– NH₂OH CellularNitrogen.
3. Nitrate metabolism of the adapted cells was investigatedundervarious conditions. In the light, nitrate was reducedand furtherassimilated, leaving insignificant amounts of nitritein themedium. In this case, consumption of nitrate was markedlyinhibitedby other forms of nitrogen (e.g., nitrite, hydroxylamine,aminoacids and ammonium salts). In the dark, nitrate was reducedas the terminal hydrogen acceptor in the oxidative breakdownof organic substances (e.g., malate) in the medium (i.e., nitraterespiration). More nitrite was accumulated in this case thanin the light. Molecular oxygen inhibited the reduction of, aswell as the growth on, nitrate in any of the above cases.
4. Theeffects on the rate of nitrate reduction (and respiratoryoxygenuptake) caused by various experimental factors (pH, nitrateconcentration, electron donors, and addition of hydroxylamine)were investigated, using the resting cells of the adapted organism.

¹ This paper was submitted to the University of Tokyo to fulfillthe requirement for the author's doctorate. ² Present Address: Botanical Institute, Kyoto University, Sakyo-ku,Kyoto. (Received February 14, 1963; )     

Growth Regulator Interactions in the Growth of the Shoot System of Avena sativa Seedling: II. THE GROWTH OF THE FIRST LEAF AND THE COLEOPTILE1

1. Studies have been made of the growth in culture medium of thecomponent parts of compositesegments excised from 3 to 7-day-oldAvena sativa seedlings and comprising portions of coleoptileand first leaf bases and various lengths of first internodetissue.
2. The effects of various concentrations of gibberellicacid (GA)and indole-3- acetic acid (IAA) alone and in combinationhavebeen studied on the growth of these organs.
3. Both GA andIAA stimulate the growth of coleoptile base tissuebut in combinationtheir joint effects are less than additive.No synergism occurs.
4. The growth of the first-leaf base is greatly stimulated byGAbut is inhibited by IAA. In combination, the stimulatoryeffectof GA (up to 1 0 p.p.m.) may be virtually eliminatedby evenlow concentrations of IAA (0.01 p.p.m.).
5. The inclusionof first internode tissue in the segments considerablyincreasesthe growth of first leaf base tissue but has no consistenteffecton the growth of coleoptile base tissue. The presenceof firstinternode tissue also greatly increases the degreeof growthstimulation invoked by GA but does not influence thedegreeof IAA inhibition. It is postulated that the first internodetissue is the source of an unknown growth factor necessary forGA action in the first leaf and potentiating the action of endogenousgibberellin.
6. Kinetin, adenine sulphate, glutarnine, glutarnicacid, asparagine,glycine, arginine, histidine, lysine, aneurin,and pyridoxinewill not simulate the effects of this unknowngrowth factorin the growth of leaf tissue. Like IAA, kinetinvirtually eliminatesthe GA stimulation of leaf growth.
7. Astudy of extracts of internode tissue in various solvents,analysedby paper partition chromatography and assayed by thegrowthof the first leaf base, has indicated the presence ofgrowthinhibitors and gibberellin-like substances but has failedtoisolate the postulated endogenous GA-synergist.
8. The implicationsof these results for growth correlations andthe hormone controlof shoot growth in Avena sativa seedlingsis discussed.

     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )     

ISOLATION OF AN INHIBITOR OF GROWTH AND ROOT FORMATION FROM PORTULACA GRANDIFLORA LEAVES

1. From leaves of Portulaca grandiflora, a substance which inhibitedthe IAA-induced elongation of Avena coleoptile sections andthe adventitious root formation of Raphanus hypocotyl cuttingswas separated by means of thin layer chromatography. It wasisolated and crystallized. 2. On paper chromatograms, this substance gave the same R_f valuesas the inhibitor from leaves of Xanthium strumarium and thatfrom leaves of Helianthus tuberosus ("heliangine"), namely,R_f0.9 in ammoniacal isopropanol, R_f 0.85 in methanol-water andR_f 0.0 in n-hexane-water. On thin layer chromatograms, however,these inhibitors were clearly separated from each other. 3. Infra-red absorption spectrum also indicated that this substanceis identical with neither xanthinin nor heliangine. ¹ Contribution No. 8 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo     

INTERACTION OF GIBBERELLIN A3, AND INORGANIC PHOSPHATE IN TOBACCO SEED GERMINATION

1. Investigation was made on the influence of inorganic phosphateupon the germination of positively photoblastic tobacco seed(Nicotiana tabacum L. var. uirginica (AGDH.) COM. "Bright Yellow")induced by GA₃, GA₃M, kinetin, red light, and ammonium saltsof various organic acids.
2. Inorganic phosphate increases theGAs-induced germination, andinhibits the germination causedby ammonium citrate, while itdoes not influence the germinationbrought about by GA₃M, kinetin,and red light.
3. The optimumpH for the GA₃-induced germination lies in the acidicpH range,indicating that the undissociated form of GA₃ is operative.The stimulatory effect of phosphate is, however, not ascribedmerely to the pH control in the mediurr. Phosphate exerts somespecific influence for which the presence of the free carboxylgroup of GAs is required.
4. The observed contrasting effectsof phosphate on the GA₃-inducedgermination (i.e., acceleration),on the one hand, and on theammonium citrate-induced germination(i.e., inhibition), onthe other, were explained by assumingthat the phosphate effectsultimately consist in acceleratingthe uptake of the carboxylicacid into the seeds.
5. GA₃M alsohas an activity of inducing the germination of tobaccoseedwithout light.

¹Present address: Department of Vegetable Crops, Universityof California, Davis, California, U.S.A. (Received March 12, 1962; )     

Oxygen enhancement of photosynthesis in Anacystis nidulans cells1

Oxygen enhanced photosynthetic ¹⁴CO₂ fixation in Anacystis nidulanscells. Results obtained under different conditions revealedthe following properties of the oxygen enhancement:

1. The enhancement was most significant at ca. 10% O₂. Furtherincrease in oxygen concentration decreased the enhancing effect.The rate under 100% O₂ was equivalent to or a little higherthan that under N₂ gas.
2. b) With the increase in CO₂ concentration,the magnitude ofthe enhancing effect decreased. No oxygen enhancementwas observedwhen the CO₂ concentration. was raised to 9,000ppm.
3. c) The enhancement was observed only at high light intensities.No enhancement was observed when the rate of photosynthesiswas limited by light intensity.
4. Ribulose 1,5-diphosphate (RuDP)carboxylase activity was demonstratedin the extract obtainedfrom A. nidulans cells. We also foundthat the RuDP carboxylaseactivity in this extract was competitivelyinhibited by oxygen.
5. Based on the above-mentioned results, the possible mechanismunderlying the observed enhancing effect of oxygen was discussed.

(Received May 10, 1976; )     

Distribution of Endogenous Indole-3-acetic Acid and Growth Inhibitor(s) in Phototropically Responding Oat Coleoptiles

The relationship between the flank growth of oat (Avena sativaL. cv. Victory) coleoptiles and the distribution of endogenousindole-3-acetic acid (IAA) and growth inhibitor(s) in the coleoptileswas studied for the second positive phototropic curvature inducedby a continuous unilateral illumination with white light (0.1W.m^–2). The phototropic curvature was caused by growthinhibition at the lighted side and growth promotion at the shadedside. Using electron capture detection gas chromatography, weanalyzed the distribution of endogenous IAA in phototropicallyresponding oat coleoptiles and found that the IAA was evenlydistributed over the lighted and shaded sides during the phototropicresponse; there was also no detectable difference in the amountsof IAA between phototropically stimulated and non-irradiatedcoleoptiles. By contrast, oat coleoptile straight-growth testresults showed that the amount of unknown acidic growth inhibitor(s),different from abscisic acid, increased in the lighted halfof the coleoptiles and decreased in the shaded half, as comparedto the amount in the non-irradiated half. These data suggestthat the phototropic curvature of oat coleoptile is inducedby a difference in lateral flank growth through a lateral gradientof endogenous growth inhibitor(s) rather than of IAA. (Received February 10, 1988; Accepted July 29, 1988)     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )     

COMPARATIVE STUDIES ON GROWTH, RESPIRATION, PHOTOSYNTHESIS AND PIGMENT CONTENT IN RHODOPSEUDOMONAS PALUSTRIS

1. Comparative studies were performed on growth, photosyntheticand respiratory activities, and pigment content in Rhodopseudomonaspalustris.
2. The growth of the organism, as influenced by variousculturalconditions such as light, aerobiosis, anaerobiosisand nutritionalfactors was investigated.
3. The respiratoryactivity of the bacterium was found to be higherin dark-growncells than in cells grown in the light. The photosyntheticactivitydid not significantly depend on the growth conditionsof theculture. Cells of younger cultures were found to be moreactivethan those of older cultures, with respect both to respirationand photosynthesis.
4. The pigment content was found to be higherin the light-growncells than in the dark-grown ones. The ratiophotosyntheticactivity/bacteriochlorophyll was significantlyhigher in thelatter than in the former.
5. Light, as well asvarious nutritional factors, was found toexert a marked accelerationon pigment formation, although ithas not yet been possibleto culture cells completely lackingin photosynthetic pigmentsand accordingly in photosyntheticactivity.

¹ Present address: Division of Dermatology and Urology, TokyoMetropolitan Hiroo Hospital, Tokyo. ² Present address: Department of Biology, Saitama University,Urawa. ³ Present address: Department of Biochemistry, School of Medicine,Yokohama University, Yokohama. ⁴ Present address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Tokyo. (Received July 23, 1961; )     

CHANGES IN RESTING POTENTIAL AND ION ABSORPTION INDUCED BY LIGHT IN A SINGLE PLANT CELL

1. Effect of light on ion absorption and resting potential of theinternodal cell of Nitella flexilis was investigated under variousconditions.
2. On illumination, the resting potential increasedby about 30mVin 10^–4 M KCl and by about 60 mV in 10^–4M NaClsolution. A similar photoelectric response was also observedin 10^–3 M KCl, 10^–2 M CaCl₂ and 5 x 10^–2 MCaCl₂ solutions, but not at all in 10^–2 M KCl solution.
3. Absorption of ions by the cell took place in parallel withthelight-induced change in resting potential.
4. Red and bluelights were very effective in increasing the restingpotential,while green light was almost ineffective. These differenteffectsof color lights were in good agreement with their effectsinincreasing the osmotic value of the cell.
5. The photoelectricresponse was not affected by phenylurethane,which, on the otherhand, strongly inhibited the light-inducedion absorption.
6. Theuptake of ions by the cell from the external medium intothevacuole is assumed to proceed in two different steps: thefirstis the process involving the ion movements across theoutermostplasmalemma, and the second is that involved in thetransportof ions through the cytoplasmic layer and tonoplast.The formerprocess is considered to be influenced by the increasein restingpotential probably caused by the light absorbed bychlorophyll.The process was, however, suggested to be independentof photosynthesis.On the other hand, the latter process issupposed to be relatedto photosynthesis. A discussion was madealong this line.

(Received July 26, 1962; )     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

LIGHT-INDUCED OXYGEN EVOLUTION CAUSED BY HYDROGEN PEROXIDE IN CATALASE-INHIBITED CHLORELLA CELLS

1. Chlorella cells and spinach chioroplasts, whose catalase activityhad been more than 90% inhibited by 10^–5 M azide, werefound to decompose H₂O₂ photochemically to liberate oxygen,indicating that H₂O₂ was used as an oxidant of the HILL reaction.
2. That, however, the observed phenomena cannot be fully accountedfor in terms of the HILL reaction with H₂O₂ was revealed bythe observation that an extract of Chiorella cells, which hadbeen completely freed from chlorophyll, also showed a light-acceleratedO2 evolution from H₂O₂ in the presence of 10^–5 M azide.This extract contained a large quantity of catalase, which seemedto have been, in some way, involved in the reaction in question.
3. The catalatic H₂O₂ decomposition caused by crystalline catalaseof mammalian liver (in the presence of 10^–5 M azide) wasnot accelerated by the effect of light.

¹ Present address: Department of Biology, Faculty of Science,Niigata University, Niigata. (Received June 4, 1961; )     

Microbiological Synthesis of Fat: THE EFFECTS OF NITROGEN LEVEL ON THE METABOLISM OF PENICILLIUM LILACINUM THOM IN A SUCROSE MEDIUM

1. A study has been made of the relationships between the synthesesof carbohydrate, protein, and fat by Penicillium lilacinum Thomin presence of different amounts of sodium nitrate us a definedsucrose salts medium.
2. Under the defined experimental conditionsincreases in the concentrationof NO^–₂ in the medium werefollowed by increases in therates at which nitrogen and sugarwere taken up by the fungus,in the quantities assimilated,and in total and protein nitrogenin the felt. These conditionsprevailed so long as unassimilatedsugar was available.
3. Mediaof lower NO^–₃ concentration (for example, 0·32or 0·64 per cent. (w/v) NaNO^–₂;) yielded feltsricher in carbohydrate than were those grown in media of higherNO^–₂; content (0·96 or 1·28 per cent. (w/v)NaNO₃ The carbohydrate content of the felts increased graduallyuntil the sugar in the medium was exhausted; carbohydrate contentthen decreased.
4. Media of lower NO^–₃; concentration weremore conduciveto fat synthesis than those of higher NO^–₃;content.

     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

INHIBITORY EFFECT OF GROWTH RETARDANTS ON THE INDUCTION OF FLOWERING IN WINTER WHEAT

1. Growth retardants, CCC, Amo-1618, Phosfon-D and B-995, appliedduring seed vernalization inhibited the ear development of winterwheat. CCC applied during green plant vernalization inhibitedflowering,but it had no appreciable effect on the final stemlength. Theinhibition by CCC was reversed by foliar applicationof gibberellin.On the other hand, CCC applied after the vernalizationperiodaffected the final stem length but not flowering.
2. Theamount of endogenous gibberellin-like substance(s) was greaterin the vernalized plant than in the non-vernalized plant. Nogibberellin-like substance was detected in the CCC-treated plant.
3. Endogenous gibberellin-like substance(s), whose biosynthesisis inhibited by some growth retardants, may play a part in thevernalization process in winter wheat.

¹Present address: National Institute of Agricultural Sciences,Nishigahara, Kitaku, Tokyo     

The Influence of Prior Water Stress and Abscisic Acid Foliar Spraying on Stomatal Responses to CO2, IAA, ABA, and Calcium in Leaves of Solanum melongena

The influence of a water stress or foliar ABA spraying pretreatmenton stomatal responses to water loss, exogenous ABA, IAA, Ca²⁺,and CO₂ were studied using excised leaves of Solanum melongena.Both pretreatments increased stomatal sensitivity of water loss,in the presence and absence of CO₂, but decreased stomatal sensitivityto exogenous ABA. CO₂ greatly reduced the effect of exogenouslyapplied ABA. IAA decreased leaf diffusion resistance for controland ABA sprayed leaves, but did not influence the LDR of previouslywater-stressed leaves. CA²⁺ did not influence LDR of any leavesof any treatments. Key words: Water stress, stomatal response, pretreatments