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

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

     

FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

STUDIES ON LACTATE CYTOCHROME C REDUCTASES OF YEAST WITH SPECIAL REFERENCE TO ELECTROPHORESIS ON POLYACRYLAMIDE GEL

1. Two lactate dehydrogenases, L(+)- and D(–)- lactate cytochromec reductase, were extracted from the baker's yeast after disintegrationof the cells by a FRENCH press. They are separated by electrophoresison polyacrylamide gel and their activities were compared bycolor density of formazan, the reduction product of nitrobluetetrazolium.
2. The ratio of L-lactate cytochrome c reductaseactivity to D-lactatecytochrome c reductase activity variedto a great extent, dependingon culture conditions. L-Lactatecytochrome c reductase waspredominant in resting cells; thereverse was the case withcells in early exponential stage ofthe growth.
3. When the cells in exponential stage of growthwere aerated withoutnitrogen source, there occurred an intensiveincrease of L-lactatecytochrome c reductase, accompanied bythe decrease of D-lactatecytochrome c reductase.
4. Effectsof inhibitors on the activity ratio of these two enzymeswereinvestigated. o-Phenanthroline, dinitrophenol, sodium azide,chloramphenicol, British antilewisite and antimycin A favored,in this order, the formation of L-lactate cytochrome c reductase.

(Received August 18, 1966; )     

LACTATE OXIDATION SYSTEM IN ACETOBACTER SUBOXYDANS, WITH SPECIAL REFERENCE TO CARBON MONOXIDE-BINDING PIGMENT

1. Lactate oxidation system was investigated in Acetobacter suboxydans,which was found to have cytochromes a and b, but no cytochromec. Haemoprotein 558 was also found to exist.
2. Carbon monoxideinhibited the lactate oxidation in the darkbut not in the light.WARBURG'S partition constant was estimatedto be 7.
3. Additionof haemoprotein 558 noticeably enhanced the oxygenuptake bythe LDH preparation, which was obtained from bacterialcellsand partially purified.
4. Haemoprotein 558 has protohaem asits prosthetic group. Notonly its absorption spectrum is reminiscentof that of peroxidase,but it also shows peroxidase-like reactivitywith some ligandswith a few exceptions.
5. Ferrohaemoprotein558 reacts with oxygen, forming, at first,a complex, whichhas its SORET absorption peak at 423 mµ.The absorptionmaximum then shifts to 417 mµ, indicatinga transformationto another compound. One of these two productsis likely tobe the oxygenated ferro-haemoprotein 558.
6. The mutual transformationbetween cytochrome B and haemoprotein558 is discussed.

(Received October 8, 1965; )     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: II. INFLUENCE OF CARBON-NITROGEN RATIO OF CULTURE MEDIUM

1. Several factors affecting sporulation of a wild yeast, Hansenulasaturnus, especially carbon sources and the carbon-nitrogenratio of sporulation medium were studied.
2. The sporulationis stimulated at a certain definite C/N ratioof glucose medium.
3. Several carbon sources such as ethanol, acetate, lactate,glycerol,succinate, glucose, gluconate and citrate are utilizedby theorganism both for growth and sporulation.
4. The numberof spores in an ascus depends on the C/N ratio ofthe medium.An increase in the ratio stimulates the yield of2-and 3-sporedasci, especially of the former. One-spored ascibecome abundantas this ratio decreases.
5. Lysine promotes sporulation in anacetate medium, and its presencein a large amount in glucosemedium also stimulates sporulation,while a small amount isinhibitory. When lysine was employedas the sole nitrogen source,most of the asci were 1-spored.
6. It is discussed that sporulationof yeast is induced by a balanceof metabolism, rather thanby one definite "sporulation substrate".

¹ Present address: Laboratory of Microbiology, Department ofAgriculture, T{circumflex}hoku University, Sendai. (Received May 23, 1961; )     

"GIGANTISM" OF CHLORELLA VULGARIS I. RELATION OF GIGANTISM TO CELL GROWTH AND DIVISION

1. The sugars which induced gigantism of Chlorella cells wereglucose,fructose, galactose, mannose, xylose and arabinose.These sugarswere utilized as respiratory substrates by thealgal cells.
2. The cellular division of Chlorella was stimulatedby glucoseand galactose, but suppressed by fructose, mannose,xylose andarabinose, while all these sugars evoked gigantism.No correlationwas found between cellular division and gigantism,
3. The photosynthetic activity of giant Chlorella varied withthesorts of sugars added. It was decreased by glucose, fructoseand mannose, but was unaffected by other sugars such as galactose,xylose and arabinose.
4. The respiratory activity of giant Chlorellacells as much higherthan that of control cells.
5. The amountsof protein-N and dry weight per unit volume of giantChlorellawere much less than those of control cells.

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM: I. OXIDATION OF SULFUR

1. Thiobacillus thiooxidans, isolated in our laboratory, was foundto oxidize sulfur, but not thiosulfate. Tetrathionate is alsooxidized slightly. Its ability to oxidize sulfur is inactivatedeven by such a mild treatment as keeping the cells in a frozenstate.
2. Inhibitory action of alcohols on the sulfur oxidationincreasesas the length of carbon chain of alcohols increases.Carboxylicacids do not inhibit the sulfur oxidation at pH abovetheirpK, while they strongly inhibit the reaction at pH belowthepK.
3. The sulfur oxidation is inhibited by cyanide, azide,diethyldithiocarbamateand carbon monoxide, and the inhibitionby carbon monoxide isnot reversed by light. These results suggestthe presence ofmetal enzymes in the sulfur oxidation system.The terminal enzymeof this reaction appears to be differentfrom the usual cytochromeoxidase.

(Received May 13, 1960; )     

Studies on Plant Growth Hormones: I. BIOLOGICAL ACTIVITIES OF 3-INDOLYLACETALDEHYDE AND 3-INDOLYLACETONITRILE

1. Two neutral plant hormones, one isolated recently from plants(3-indolylacetonitrile) and the other (3-indolylacetaldehyde)reported to be present in plants, are avaible as pure syntheticcompounds for investigation of their biological activities.This paper is mainly concerned with their effects on cellelongationin the Avena coleoptile
2. 3-Indolylacetaldehyde is considerablyless active than 3-indolylaceticacid in the Avena straight-growthtest; for example, a 1.0 mg./l.solution of the aldehyde showsan activity equivalent to thatof a 0.1 mg./l. solution of theacid
3. An acidic substance is produced in solutions of the aldehydeduring the period of assay. In some experiments it accountsfor all of the activity shown by the aldehyde solutions, onthe assumption that it is 3-indoylacetic acid, and in otherexperiments it shows a greater activity than that of the aldehydesolutions from which it was obtained. Therefore, it is concludedthat 3-indolylacetaldehyde, itself is either inactive or inhibitory.Acid production in aldehyde solutions in vitro is much lower,a fact which suggests that there is enzymatic oxidation of aldehydeto acid in the presence of coleoptiles.
4. The activities of3-indolylacetaldehyde in the pea test andin root-inhibitionand of 3-indolylacetone in the straight-growthtest are brieflyreported.
5. 3-Indolylacetonitrile is considerably more activethan 3-indolylaceaticacid in the Avena straight-growth test;for erample, a 0.1 mg./l.solution of the nitrile shows an activityequivalent to a 1.0mg./l. solution of the acid. The inhibitoryeffect at concentrationsabove 1.0–10.0 mg./l. is lesswith the nitrile than withthe acid.
6. There is negligible productionof acid in solutions of the nitrileboth in vitro and in thepresence of Avena coleoptiles at temperaturesranging from –18?to 25? C. for varying lengths of time.The possibility of enzymaticconvesion of nitrile to acid insidethe cells of the coleoptileis discussed
7. The activities of 3-indolylacetamide and of 2:4-dichlorophenoxyaceticacid and the corresponding nitrile are considered in this connexion
8. The nitrile is destroyed by treatment with alkali but notbyacid. In the light of these results, it is desirable to re-examineprevious work on identification of auxins in plants by theiracid and alkali sensitivity. Evidence for the existence of thenitrile in a number of other plants is presented.

     

The Metabolism of Glucose and Acetate in Aspergillus niger

1. The metabolism of a citric-acid-forming strain of A. nigerwhengrown on a glucose-acetate medium has been investigated.
2. Acetate greatly accelerated the rate of utilization of glucose.
3. Citric acid production from glucose was much increased bythepresence of acetate.
4. The formation of oxalate from glucose-acetatecultures was muchless than from acetate alone.
5. In some cultureslarge amounts of glucose and acetate were consumedbut no acidicproducts were formed.

     

Further Studies on the Acid Metabolism of Aspergillus niger: THE FORMATION AND UTILIZATION OF OXALIC ACID

1. Some recent works on the formation of oxalic acid by variousfungi are critically considered.
2. The present work deals withthe role of oxalic acid in the metabolismof Aspergillus niger.
3. When glucose solutions were supplied to preformed mats ofthefungus oxalic acid accumulated, attaining an equilibriumlevelwhich was not exceeded despite the presence of a considerableconcentration of glucose.
4. When the glucose supplies were depletedthe oxalic acid concentrationfell steeply to a low level.
5. Theconcentration of oxalic acid was dependent on the glucoseconcentration.In three separate series of experiments it wasshown that theoxalic acid concentration diminished with increasingglucoseconcentration.
6. Similar results were obtained when the cultureswere rearedfrom spores on culture solutions with the normalamounts ofnutrient salts but different glucose concentrations.
7. In all cases the CO₂ output increased with the glucose concentration.
8. When cultures were supplied with glucose+oxalic acid, theconcentrationof the latter fell steeply to the equilibriumlevel attainedon glucose only. In a culture receiving glucose+oxalicacid,with the oxalic acid concentration somewhat below thenormalequilibrium concentration, the formation of oxalic acidfromthe glucose ceased as soon as the equilibrium level hadbeenattained.
9. When 1 per cent. oxalic acid only was suppliedto the fungusthe concentration gradually diminished to a lowlevel. When3 per cent. oxalic acid was supplied the rate ofacid utilizationsoon fell to low value.
10. In several experimentsit was shown that the rate of CO₂ outputwas higher from culturessupplied with glucose+excess oxalicacid than from culturessupplied with glucose only.
11. The rate of oxalic acid carbonloss was always below that ofthe CO₂ carbon output both incultures supplied with oxalicacid only and in cultures receivingglucose+oxalic acid.
12. The cultures were incapable of utilizingneutral sodium oxalateand the presence of this substance hadno effecft on the ofCO₂ output.
13. The results indicate thatthe utilization of oxalic acid isassociated with the liberationof at least an equivalent amountof CO₂.
14. It is suggested thatthe utilization of oxalic acid is promotedby the presence ofglucose, thus accounting for the lower oxalicacid concentrationsand higher rates of CO₂ output of cultureswith higher glucoseconcentrations.

     

"GIGANTISM" OF CHLORELLA VULGARIS I. MECHANISM OF INDUCTION OF CIGANTISM

1. Based on the microscopic observations, two stages, "giant cellstage" and the subsequent "palmelloid body stage", were distinguishedin the process of formation of giant Chlorella induced by theaddition of sugars. The "giant cell" is much larger in sizethan the control cell, but the other morphological featuresare the same as those of the latter. The "palmelloid body" isa form composed of many conjoined autospores.
2. When a highconcentration of glucose was maintained in the medium,gigantismwas also maintained. Under this condition, the algashows acyclic transformation between "giant cell" and "palmelloidbody"without returning to the small single cells.
3. Large amountsof carbohydrate composed of hexose were foundto be accumulatedin the giant algal cells, and it was inferredthat this carbohydrateaccumulation causes greater enlargementof cell volume as comparedwith control cells.
4. Uronic acids, which were found to be absentin the control cells,were formed and lost in the cells culturedin the glucose mediumin parallel with the appearance and disappearanceof gigantism.
5. Pectic substances, from which uronic acids areconsidered tobe derived during the extraction procedure, werefound to bepresent only in giant Chlorella.
6. The conjoinedautospores in giant Chlorella (at the palmelloidbody stage)were separated to some extent by the addition ofEDTA, and theresulting cells were similar to control Chlorellacells.
7. Basedon these results it was inferred that inductive formationofthe pectic substances is causally related with the appearanceof "palmelloid body".

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

Polyphenoloxidase and the Respiration of Ivy Leaves

1. Polyphenoloxidase is present in ivy leaves but not in thoseof Aucuba or Euonymus.
2. Respiration of intact ivy leaves wasmarkedly stimulated bycatechol (R.Q. approximately=I), gallicacid, caffeic acid,and dihydroxyphenylalanine. The stimulationwas not relatedto injury as far as could be detected and wasnot followed byinhibition. The extra oxygen consumption representsa many timesrepeated oxidation of the amount of catechol supplied.
3. The effect of catechol on the respiration of Aucuba and Euonymusleaves was very small.
4. Cupferron and phenylthiourea, whichinhibit polyphenoloxidasein vitro, nevertheless increased respirationwhen administeredto leaves through the petiole. On the otherhand, when appliedby infiltration, cupferron did cause inhibition,but in Aucubaand Euonymus as much as in ivy.

     

PHOTOCHEMICAL REDUCTION AND OXIDATION OF MENADIONE (VITAMIN K3) IN CHLOROPLASTS

1. Menadione (vitamin K³) was found to be completely reduced byilluminated spinach chloroplasts under highly anaerobic conditionand in the presence of ethylenediamine tetraacetate (EDTA) inthe reaction mixture. This photoreductive reaction is sensitivetoward heat-treatment and inhibited by 2?10^-3M hydroxylamine.
2. In the presence of oxygen, the reduced form of menadione israpidly photooxidized by chloroplasts. This photooxidative activityalso is suppressed by heat-treatment but not inhibited by hydroxylamine.
3. Dyes which are inefficient as HILL oxidants such as thionineand methylene blue were found to be readily reduced by illuminatedchloroplasts, if the experimental conditions were appropriateto prevent the reoxidation of the photoreduced dyes; i.e., exhaustiveremoval of oxygen and the addition of EDTA in the reaction mixture.Menadione was found to accelerate the HILL reaction with thesedyes as oxidant under such experimental conditions.
4. In thepresence of molecular oxygen in the reaction mixture,menadionewas found to inhibit the HILL reaction with 2,6–dichlorophenolindophenol as oxidant, while the reaction rate was little influencedin high anaerobiosis.
5. These findings are explained by theintermediary oxidation and(photo-) reduction of menadione asan intermediary hydrogencarrier, and by the trends toward rapidphotooxidation of reducedmenadione.

(Received July 2, 1960; )     

PHOTOOXIDATION REACTIONS BY CHLOROPLASTS SOLUBILIZED WITH SURFACE-ACTIVE AGENTS

1. Solubilization of chioroplasts with a mixture of 1 per centDuponol C and 1 per cent Span 80 (3: 1) caused a destructionof activity in the HILL reaction, but the treatment broughtabout an increase by about 60 per cent in the rate of ascorbatephotooxidation in the presence of DPIP. Heating the broken chloroplastscaused a marked decrease in the photooxidation activity. Byadding surface- active agents to the boiled preparation, theactivity was restored up to almost 80 per cent of the originallevel.
2. With colloidal suspensions of isolated chiorophylls,ascorbatewas only slightly photooxidized in the presence ofDPIP. Byaddi tion of the surface-active agents, the activitywas greatlyenhanced.
3. Dependency of the photooxidation bywhole and solubilized chloroplastsand isolated chlorophylla on the presence of DPIP was examined.DPIP can serve as anintermediate electron carrier in solubilizedchloroplasts aswell as in whole chloroplasts.
4. Effect of o-phenanthrolineon ascorbate photooxidation by thesethree preparations wastested. With solubilized chloroplastsand isolated chlorophylls,the addition of the inhibitor hadno influence on their ascorbatephotooxidation either in thepresence or absence of DPIP.
5. Treatmentof whole chloroplasts with the surface-active agentsinducedan activity of photooxidation of cytochrome c. The electron-flowpattern for the photooxidation of ascorbate by whole and solubilizedchloroplasts was briefly discussed.

¹ Contribution No. 130 from the Department of Biology, Facultyof Science, Kyushu University. Aided in part by Grant-in-Aidfor Fundamental Scientific Research from the Ministry of Education. (Received August 23, 1962; )     

STUDIES ON CHLOROPHYLLASE OF CHLORELLA PROTOTHECOIDESI. ENZYMATIC PHYTYLATION OF METHYL CHLOROPHYLLIDE

1. The relation between chlorophyll content and the hydrolyticactivity of chlorophyllase in Chlorella protothecoides was examined.An increase in the activity was parallel to that in chlorophyllcontent during the development of green colouration, or greeningcourse, in the bleached cells. The activity sharply declinedand a parallel disappearance of chlorophyll was also found duringbleaching of the green cells.
2. A partially purified water-solublepreparation of chlorophyllasewas obtained by n-butanol treatmentand fractionation with coldacetone. It showed high activityand hydrolyzed 2 mg chlorophylla per hr per mg protein.
3. Forseparation and identification of the pigments concernedin thechlorophyllase reaction, a new solvent system of paperchromatographywas introduced.
4. When methyl chlorophyllide a and phytol wereincubated withthe enzyme, two products were formed. By comparisonwith theRf values of isolated pure substances, one was identifiedaschorophyll a and the other as chlorophyllide a. This enzymedid not catalyze the phytylation of free chlorophyllide a, butit had the ability to attach phytol to methyl chlorophyllidea. The final step in the biosynthesis of chlorophyll a is brieflydiscussed.

¹ Contribution No. 158 from the Department of Biology, Facultyof Science, Kyushu University. Supported in part by a grant-in-aidfor Fundamental Scientific Research from the Ministry of Education.     

FLAVOPROTEINS IN THE LEAVES OF HIGHER PLANTS CATALYZING THE OXIDATION OF L-GLUTAMATE

1. Two forms of enzyme capable of catalyzing the oxidation of L-glutamate(and L-aspartate) were isolated from the leaves of spinach andseparated from each other by column-chromatographic purificationon calcium phosphate and anion exchangers. They were distinguishedas GD₁ (L-glutamate dehydrogenase 1) and GD₂ (L-glutamate dehydrogenase2). The purification procedures and some fundamental propertiesof the partially purified enzymes were investigated.
2. It wasdiscovered that the enzymes did not require any cofactor,ie., neither dialysis nor precipitation with ammonium sulfatecaused a fall in enzyme activities and the addition of DPN andTPN to the reaction mixture did not accelerate the reactionrate
3. From the results of spectroscopic investigation GD₁ andGD₂were shown to be flavoproteins, although their prostheticgrouphas not yet been identified The activity of GD₁ was enhancedby the addition of FAD or FMN, while GD₂ was not acceleratedby these factors.
4. The characteristics of the two enzymes includingsubstrate specificity,MICHAELIS constant, optimum pH of thereaction and specificityfor electron acceptors were compared.
5. From the stoichiometric study of the oxidation of L-glutamatewith these enzymes, it was confirmed that the reaction is representedby the following equation: L-glutamate+oxidized dye+h₂o
6. Among various inhibitors tested,molecular oxygen which couldfunction as electron acceptor ofL-glutamate oxidation in thepresence of GD₁ was found to causea strong inhibition uponthe same reaction with TTC as el acceptor.The inhibition wasconfirmed to be due to hydrogen peroxideproduced as a resultof the aerobic oxidation of L-glutamate.

(Received July 25, 1962; )     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

Studies on Plant Growth Hormones: II. FURTHER BIOLOGICAL PROPERTIES OF 3-INDOLYLACETONITRILE

1. 3-Indolylacetonitrile is more active than 3-indolylacetic acidin the Avena straight-growth test, but less active in the Avenacurvature test at comparable concentrations. Reasons for thisare discussed, and results of previous work on plant extractsusing the curvature test as a means of assay are considered.
2. Transport of both the acid and the nitrile is polar, fromapexto base of the coleoptile. The nitrile can reach the growingcells as easily, and possibly more easily, than the acid. Thesignificance of these findings for a theory on the mechanismof action of the nitrile is discussed.
3. The nitrile is inactivein the pea curvature test and straight-growthof pea stem sectionsexcept at high concentrations. It is alsoinactive or only slightlyactive in lateral bud inhibition,root initiation, and petioleabscission at the concentrationstested.
4. It is less activethan the acid in root inhibition in cress,but approximatelyas active in Avena. It is approximately asactive as the acidin parthenocarpic fruit development, andinitiation of cambialactivity.
5. The significance of these results is discussed.

     

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     

VARIATION OF WATER CONTENT IN HELIX ASPERSA MULLER IN A NATURAL ENVIRONMENT

1. Daily and seasonal variations occur in the water content ofHelix aspersa Müller.
2. The water content is not constantthroughout 24 hours but fluctuatesby 2 to 10%. There is noapparent daily rhythm.
3. Humidity has an effect on the watercontent. Falls in relativehumidity to 60% or less, triggera decrease of the snails' hydration,over a range of temperatures,as well as a brief pause in activity.
4. Only external climaticconditions produce noticeable fluctuationsof water content,and they more particularly affect juvenileswhich undergo greaterevaporo-transpiration.
5. A seasonal rhythm in water contentexists in Helix aspersa,with maxima during spring and autumnand minima in June andJanuary.
6. The water content of juvenilesis always superior to that ofthe adults.
7. The water contentaffects the duration of the snail's activephase.

(Received 22 September 1988; accepted 5 February 1989)