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; )     

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; )     

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)     

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)     

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; )     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Effects of Metal Chelators on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after being detachedand kept in the dark was studied in terms of the loss of chlorophyll.Chlorophyll was more rapidly degraded in maize than hydrangeaduring the incubation period in the dark. The loss of chlorophyllin the dark was effectively inhibited in both plants by ,'-dipyridyland o-phenanthroline at concentrations between 0.01 and 0.1mM. Three other chelators of iron produced lesser inhibitionand only at higher concentrations. EDTA prevented the loss ofchlorophyll in maize leaves at concentrations above 10 mM, butdid not do so in hydrangea leaves. Detached leaves floated on EDTA, ,'-dipyridyl or o-phenanthrolinesolutions and exposed to light exhibited a marked bleaching.The bleaching was partially inhibited by applying ascorbic acid. (Received December 26, 1981; Accepted October 18, 1982)     

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)     

HORMONAL REGULATION ON THE INDUCTION OF {alpha}-AMYLASE ISOZYMES IN THE EMBRYO-LESS ENDOSPERM OF BARLEY

The -amylase induced by helminthosporol and gibberellic acidin the embryo-less endosperm of barley was separated into thethree fractions, 1, 2 and 3, by DEAE-cellulose column chromatography.A maximum amount of the 2. was induced by gibberellic acid andthat of the as by helminthosporol. After rechromatography, the2 induced by gibberellic acid and the as induced by helminthosporolshowed their respective single bands in an electrophoresis agargel zymogram. On the other hand, the ai induced by helminthosporoland gibberellic acid showed three bands. Dihydrohelminthosporic acid, a derivative of helminthosporol,induced the same isozymes as helminthosporol did. (Received May 8, 1967; )     

Purification and properties of {delta}-aminolevulinic acid dehydratase from radish cotyledons

-Aminolevulinic acid dehydratase (5-aminolevulinate hydro-lyase,EC 4.2.1.24 [EC] ) was purified from greening radish cotyledons. Thefinal product was homogeneous on polyacrylamide disc gel electrophoresisand had a molecular weight, estimated by gel filtration, of282,000 daltons. The enzyme seems to require magnesium ion aswell as sulfhydryl compounds for maximum activity. EDTA anda low concentration of zinc ion markedly inhibited the activity.The optimum pH was 8.0; the Km value for -aminolevulinic acidwas 3.85?10^–4M. Levulinic acid was a competitive inhibitorof the enzyme, with a Ki of 2.14?10^–4M. These propertieswere compared with those of microorganism and animal -aminolevulinicacid dehydratases. (Received November 22, 1976; )     

A Mutation Leading to Constitutive Expression of High Sexual Activities in the Yeast Saccharomyces cerevisiae

An a mating type mutant of the yeast Saccharomyces cerevisiaewhich expressed high sexual activities during vegetative growthwas isolated and characterized. Its constitutive sexual agglutinabilitywas higher than the sexual agglutinability of its parental straininduced by pheromone. It produced a pheromone and -pheromone-inactivatingsubstances in larger amounts than its parental strain. It alsoproduced large pear-shaped cells (shmooed cells) without pheromone,was more sensitive to pheromone, and grew vegetatively moreslowly than its parental strain. When the mutant was crossedto a wild type strain isogenic with the parental strain, amating type segregants with high constitutive sexual agglutinabilityshowed self-shmooing. However, in a mating type segregants self-shmooingwas not observed regardless of the degree of their sexual agglutinability.The cross between a and segregants, both of which carried themutation, had higher frequency of zygote formation than thecrosses between a and cells one of which or both of which wereof wild type. (Received September 9, 1985; Accepted February 8, 1986)     

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)     

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)     

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; )     

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; )     

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)     

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)