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; )     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

SPLITTING OF {varepsilon}-CAPROLACTAM AND OTHER LACTAMS BY BACTERIA

-Caprolactam-utilizing bacteria split -caprolactam, -valerolactamand -butyrolactam, and produce the -amino acids correspondingto them. This activity is lost when cells are grown on 6-amino-caproicacid or ammonium adipate, and reappears when cells are incubatedwith either -caprolactam or -valerolactam as the sole majororganic nutrient. Chloramphenicol inhibits this adaptation.The enzyme splitting those lactams is one and the same. It maybe called "lactam-splitting enzyme". But attempts to demonstratethe enzymic activity in a cell-free system has not yet beensuccessful. (Received September 9, 1965; )     

BACTERIAL BREAKDOWN OF {varepsilon}-CAPROLACTAM AND ITS CYCLIC OLIGOMERS

Eleven different types of bacteria were isolated which werecapable of growing on -caprolactam, the monomeric material fornylon 6 polyamide, as the sole source of both carbon and nitrogen. The optimal concentration of -caprolactam for the bacterialgrowth was about 0.6% in a synthetic liquid medium enrichedwith a small amount of yeast extract. The bacterial strains grew also on -butyrolactam, -valerolactamand the -amino acids corresponding to these lactams and -caprolactam.Ammonium adipate was a good substrate for the growth of allthe strains. One strain of Corynebacterium aurantiacum was found to be capableof utilizing cyclic and linear oligomers of 6-aminocaproic acidwith an exception of cyclic dimer. The strains of corynebacteria required vitamin B₁ for growth. Metabolism of -caprolactam and related compounds is discussedbriefly. (Received September 9, 1965; )     

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 Assessment of Gibberellin Structure-activity Relationships

Information on the biological activities of gibberellins (GAs)in the barley aleurone, Tangin-bozu dwarf rice, dwarf pea, lettucehypocotyl and cucumber hypocotyl bioassays is reviewed and discussedin the context of GA structure-activity relationships. The barley aleurone bioassay exhibits a limited response toGAs and it is suggested that this may be because the aleuronecells are able to carry out few GA interconversions. Consequentlyactivity is determined by the degree of compatibility betweenthe GAs and a receptor site. In this assay high biological activityis associated with GAs having a 3ß-hydroxy--lactonestructure. This activity is substantially enhanced by the additionalpresence of a 13-hydroxyl group. The substitution of a -lactoneor a -lactol for a -lactone results in reduced activity while3ß,13-dihydroxy GAs with either 20-carboxyl or 20-methylfunctions are completely inactive. The Tanginbozu dwarf ricebioassay responds to many more GAs than the barley aleuronesystem possibly because the rice seedlings can carry out extensiveGA interconversions. Under these circumstances GAs that areinactive per se can be metabolically converted to active forms.There is no interaction between the 3ß- and 13-hydroxyfunctions of GA molecules in the rice assay. Activity appearsto be determined by the degree oxidation of the C-20 group.The order of activity is usually -lactone > -lactol >-lactone > methyl > carboxyl. It is suggested this mayindicate that in rice seedlings C₂₀-GAs are converted to C₁₉-GAsvia a Baeyer-Villiger type oxidation. Activity in the dwarfpea bioassay is dependent upon GAs possessing both 3ß-and 13-hydroxyl groups and is again related to the state ofoxidation at the C-20 locus. In the lettuce bioassay activityis restricted to GAs with a -lactone function. In some instancesa -lactone, but not a -lactol, can substitute effectively. Thismay imply that the applied C₂₀-GAs are not converted to C₁₉-GAsand that the response to the -lactone results from the six-memberedring mimicking the -lactone at the receptor site. Only GAs havinga 19,10 or a 19,20 lactonic bridge show substantial activityin the cucumber bioassay. The additional presence of eithera 12- or a 13-hydroxyl group severely reduces activity.     

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)     

Nucleoside Triphosphate(NTP)-Binding Proteins and Endogenous ADP-ribosyl Transferase in Neurospora crassa

Nucleoside triphosphate(NTP)-binding proteins were detectedin the crude extract of mycelia of Neurospora crassa, whichwas treated with 1% Lubrol PX and fractionated by gel filtration.Protein fractions showing the capacity to bind [³⁵S]ATPS or[³⁵S]GTPS were designated as AGN1 to 6. The binding of [³⁵S]ATPSor [³⁵S]GTPS was prevented in the presence of 0.1 mM ATP orGTP except that in fractions AGN1 and 2, the presence of GTPstimulated the binding of [³⁵S] ATPS to ATP(NTP)-binding proteins.ATP or GTP was 1 to 2 orders of magnitude more effective thanCTP or UTP in preventing the binding of [³⁵S]GTPS in AGN1, 2and 5. Among these fractions AGN1, 2, 5 and 6 showed activityto hydrolyze 1 nM [–³²P]ATP or [–³²P]GTP. NTP-bindingproteins bound with [³⁵S]ATPS or [³⁵S]GTPS had lower apparentmolecular weights than the same proteins without bound nucleotide.Proteins bound with [³⁵S]ATPS or [³⁵S]GTPS and those [³²P]ADP-ribosylatedby endogenous ADP-ribosyl transferase in each fraction wereanalyzed by SDS-PAGE. About 20 species of ATP or ATP-GTP-bindingproteins were detected, several of which were ADP-ribosylated.The binding of [³⁵S]ATPS or [³⁵S]GTPS to NTP-binding proteinswas confirmed by the comparison of non-boiled and boiled samplesimmediately before loading to SDS-PAGE. ATP, GTP, CTP or UTPat the concentration of 0.1 mM effectively removed [³³S]ATPSor [³⁵S]GTPS bound to NTP-binding proteins. (Received December 10, 1990; Accepted April 18, 1991)     

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)     

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)     

Pumpkin (Cucurbita sp.) seed globulin VI. Proteolytic activities appearing in germinating cotyledons

N--benzoyl D,L-arginine p-nitroanilide (BAPA), leucine p-nitroanilide(LPA) and casein hydrolytic activities were assayed in germinatingcotyledons. BAPA hydrolytic activity was not detected in dryseeds, but increased rapidly from 1 to 4 days of germinationand then decreased. LPA and casein hydrolytic activities weredetected in dry seeds and increased from 2 to 4 days. Caseinhydrolytic activity decreased faster than the other two activities. BAPA hydrolytic enzyme was partially purified. It was inhibitedby p-chloromercuribenzoate and activated by ß-mercaptoethanoland dithiothreitol, but was not affected by EDTA, phenylmethylsulfonylfluoride, pumpkin trypsin inhibitor and several divalent cations.It had no ability to hydrolyze globulin or the chain to produce F_ß or smaller polypeptides,respectively, which was referred to as proteolytic activityI in the preceding paper (14), but released small peptides andamino acids from the chain and F_ß. However, it wasdifferent from proteolytic enzyme II which was present in dryseeds and inhibited by EDTA (14). Pumpkin trypsin inhibitor was purified. Its molecular weightwas estimated to be 10,500 by gel filtration. It did not inhibitthe BAPA hydrolytic enzyme. Both proteolytic activities I andII were also not reduced by the inhibitor (14). The inhibitoryactivity decreased gradually during germination. (Received November 9, 1979; )     

Discrimination and characterization of photosystem I-and photosystem II-induced 515-nm absorbance changes in chloroplasts II. Separate local fields in thylakoids indicated by differential responses of the 515-nm absorbance change

Flash-induced 515-nm and 475-nm absorbance changes in spinachchloroplasts were investigated in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU). DCMU reduced the magnitude of the 515-nmabsorbance change by half and almost completely diminished theabsorbance change at 475-nm. The reduction of the 475-nm absorbancechange paralleled the inhibition of the photosystem II (PS II)light reaction. When chloroplasts were illuminated with red or far-red light,the ratio of A₅₁₅/A₄₇₅ changed depending on the photosystemactivated. Wide variations in the A₅₁₅/A₄₇₅ ratio observed insubchloroplast particle preparations were probably due to theenrichment and activation of one of the photosystems. We suggest that the photosynthetic pigments in the thylakoidmembrane are heterogeneously distributed, and chlorophyll bmolecules that may be responsible for the 475- nm absorbancechange are affected by the local field formed by the PS II lightreaction. On the other hand, an electric field due to the PSI reaction probably induced the absorbance change at 515-nm (Received February 24, 1978; )     

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

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

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; )     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Temperature-Dependent Inhibitive Actions of {alpha},{alpha}'-Dipyridyl and Cycloheximide on the Senescence of Maize Leaves

Temperature-dependent inhibitive actions of ,'-dipyridyl andcycloheximide on the senescence of maize leaves were studied.,'-Dipyridyl effectively inhibited the loss of chlorophyll at25?C but not at 35?C. Gycloheximide was highly effective inpreserving chlorophyll at both of 25 and 35?C. Spectral analysisof senescent leaves at 35?C in ,'-dipyridyl showed simultaneousbleaching the carotenoid and chlorophyll. (Received February 20, 1984; Accepted June 14, 1984)     

Ammonia Regulation of Intermediary Metabolism in Photosynthesizing and Respiring Chlorella pyrenoidosa: Comparative Effects of Methylamine

The natural ¹³C abundance (¹³C value) of the field-grown leguminousplants (soybean, kidney bean, pea, azuki bean, mung bean, peanutand cowpea) was investigated by mass spectrometry with a precisionbetter than %0.2 for ¹³C. Among organs of premature plants,the leaves had the most negative values, and the nodules generallyhad the least negative values, and other organs, fruits, stemsand roots, showed intermediate values. In the soybeans so farinvestigated, the grains of nodulating plants exhibited higher¹³C values than nonnodulating lines. The ¹³C values of the grainsvaried depending on the species: peanuts showed the most negativevalues. Possible causes underlying these variations are discussed. (Received March 2, 1983; Accepted May 27, 1983)