Metabolic conversion of N-methyl carbon of {gamma}-glutamylmethylamide to caffeine in tea plants

Tea seedlings were treated with ¹⁴C-methylamine to cause synthesisof ¹⁴C--glutamylmethylamide (N-methyl-¹⁴C). The metabolic conversionof -glutamylmethylamide was studied by tracing ¹⁴C. ¹⁴C--Glutamylmethylamide (N-methyl-¹⁴C) translocated from rootsand cotyledons to shoots of tea seedlings, was converted almostentirely into caffeine. Conversion was greater in light-exposedsamples. For those grown in the dark, the converted amount didnot correspond to the total caffeine produced. More ¹⁴C--glutamylmethylamidewas present in stems than in leaves, but with ¹⁴C-caffeine,the opposite was found. When ¹⁴C--glutamylmethylamide or ¹⁴C-methylamine was appliedto leaf disks, ¹⁴C-caffeine was biosynthesized from both substances. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received September 25, 1971; )     

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)     

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)     

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)     

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; )     

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; )     

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)     

Purification and properties of soluble cytochrome b-560 from spinach leaves

A b-type cytochrome having an -band at 560 nm was isolated fromspinach leaves (Spinacia oleracea). A method is described forpreparing this cytochrome, cytochrome b-560 (spinach), in apurified state. The cytochrome has, in its reduced state, absorption bands at560 nm (), 530 nm (ß) and 427 nm (); and in the oxidizedstate at 562 nm (), 529 nm (ß) and 417 nm (). Thepyridine ferro-haemochrome prepared from cytochrome b-560 hadan -band at 556.5 nm, indicating the protohaem-nature of theprosthetic group. The cytochrome has an oxidation-reduction potential (E'⁰) of+0.13V at pH 7.0, as measured using the ferri-ferro oxalate system. The cytochrome is rapidly reduced on illumination with red orfar-red light in the presence of spinach chloroplasts and isoxidized at a slower rate in the dark. This photoreduction isinhibited by 1x10^–6 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The molecular weight of the cytochrome is 30,000 asestimated by the dextran gel filtration method. (Received December 3, 1971; )     

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; )     

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)     

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; )     

Discovery of Natural Photosynthesis using Zn-Containing Bacteriochlorophyll in an Aerobic Bacterium Acidiphilium rubrum

We discovered natural photosynthesis using Zn-containing bacteriochlorophyll in an acidophilic bacterium Acidiphilium rubrum. Chemical analysisof the cell extracts gave a 13 : 2 :1 molar ratio of Zn-bacteriochlorophyll : Mg-bacteriochlorophyll : bacteriopheophytin . Most of thepigments are associated with fully active reaction center andlight-harvesting complexes analogous to those in purple photosyntheticbacteria. The finding indicates an unexpectedly wide variabilityof photosynthesis. ⁷Present address: Department of Ecological Engineering, ToyohashiUniversity of Technology, Tenpaku-cho, Toyohashi, 441 Japan     

The Incorporation of C14-labelled Substrates into the Amino-acids of Groundnut Plants (Arachis hypogaea)

The amino-acid metabolism of groundnut plants has been studiedwith special reference to -methyleneglutamic acid (-MGA) andy-methyleneglutamine (-MG), constituents not found in the greatmajority of plant species. The sequence in which C¹⁴ from radioactive C¹⁴-carbon dioxideenters the amino-acids of leaves was determined. The patternof labelling was very similar to that found for leaves of otherspecies. The metabolic relationships existing between photosynthesisand amino-acid synthesis therefore do not seem to be affectedby the large quantities of -MGA and -MG present in the leaves.-MGA and -MG only gained traces of radioactivity. Experiments designed to study the incorporation of C¹⁴ fromuniformly labelled C¹⁴-alanine into the amino-acids of roots,immature leaves and cotyledons of seedlings and young plantsindicated that the main site of synthesis of -MGA was the cotyledons. Various C^I4-labelled substrates were fed to germinating seedsand, after a period of growth, the specific activities of theamino-acids of seedlings receiving different treatments weredetermined. Comparison of the specific activities enabled certaindeductions to be made concerning the probable biosynthetic pathwaysleading to -MGA and -MG. The results were consistent with theintact incorporation of pyruvate molecules, or another related3-carbon-atom containing compound, into -MGA.     

Changes in the Levels of Free Amino Acids in Various Regions of Cuscuta during Growth

The angiospermous plant parasite Cuscuta derives reduced carbonand nitrogen compounds primarily from its host. Free amino acidsalong Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15cm, and 15 to 30 cm, which in a broad sense represent the regionof cell division, cell elongation and differentiation and vasculartissue differentiation respectively, were quantitatively estimated.The free amino acid content was the highest in the 0 to 5 cmregion and progressively decreased along the posterior regionsof the vine. The haustorial region showed the lowest contentof free amino acids. In general, the free amino acid contentin samples collected at 7 p.m. was found to be higher than thatin the samples collected at 7 a.m. Three basic amino acids,histidine, the uncommon amino acid -hydroxyarginine, and arginineconstituted more than 50% of the total free amino acids in allthe zones studied except the haustorial region. Aspartic acidand glutamic acid constituted the major portion in the acidicand neutral fraction of amino acids. Glutamine, asparagine,threonine, and serine were eluted together and occurred in substantialamounts. -Hydroxyarginine constituted the largest fraction inthe cut end exudate of Cuscuta and presumably appeared to bethe major form of transport amino acid. -Hydroxyarginine wasalso a major constituent of the basic amino acids in Cuscutavines parasitizing host plants from widely separated families,suggesting that this amino acid is a biosynthetic product ofthe parasite rather than that of the hosts. Also, U-¹⁴C argininewas converted to -hydroxyarginine by cut Cuscuta vines, suggestingthat -hydroxyarginine is synthesized de novo from arginine byCuscuta. (Received March 30, 1987; Accepted June 7, 1988)     

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; )     

Effect of Growth Temperature on Phospholipid and Fatty Acid Compositions in a Psychrotrophic Bacterium, Pseudomonas sp. Strain E-3

The effect of growth temperature on the compositions of phospholipidsand fatty acids of the psychrotrophic bacterium Pseudomonassp. strain E-3 was studied. The composition of phospholipids(phosphatidylethanolamine, phosphatidylglycerol and cardiolipin)did not differ significantly in cells grown at 5?, 15?, and30?C. Phosphatidylethanolamine (PE) was the most abundant, amountingto over 70% of total phospholipids. When the growth temperaturewas lowered, palmitoleic [16:1(9)] and cis-vaccenic [18:1(11)]acids increased at the expense of palmitic (16:0) acid, especiallyin PE. In vivo experiments using [l-¹⁴C]16:0 showed that incorporationof radioactivity into 16:1(9) was oxygen-dependent and cerulenin-insensitive,and incorporation into 18:1(11) was cerulenin-sensitive. Theseresults suggest that 16:0 was aerobically desaturated to 16:1(9)and subsequently elongated to 18:1(11). Desaturation activityof 16:0 in the cells grown at 25?C was enhanced by loweringthe assay temperature. (Received February 12, 1987; Accepted July 10, 1987)     

The Role of Glutamate Decarboxylase and {gamma}-Aminobutyric Acid in Germinating Barley

The variations in glutamate decarboxylase activity and in glutamicacid and -ABA concentration have been measured in barley embryosduring the uptake of water and in the roots and shoots for upto 6 days of growth. Glutamate decarboxylase activity was relativelysteady in the embryos during soaking but rose rapidly once growthbegan. This development paralleled an increase in the concentrationof glutamic acid in both roots and shoots at a time when theconcentration of -ABA was falling. During soaking in aeratedwater, the -ABA content of the embryos rose for 36 h, at whichpoint it accounted for 35 per cent of the soluble amino acids.-ABA was found to be a major free amino acid in roots but notin shoots. Experiments in vivo involving ¹⁴C-labelled glutamicacid and -ABA indicated that carbon from -ABA passed very rapidlyinto the citric-acid cycle intermediates and also that, throughoutthe period studied, -ABA was formed from glutamic acid despitethe alterations in relative concentrations of these amino acidsin the growing tissue.     

Xyloglucan and r{beta}-D-Glucan in Cell Walls of Rice Seedlings

Cell walls of 4-day old rice seedlings were extracted successivelywith ammonium oxalate-oxalic acid, 4% KOH and 24% KOH. A rß-D-glucanpreparation and a xyloglucan preparation were isolated fromthe 4% KOH extract and 24% KOH extract, respectively. Methylationanalysis and enzymic degradation studies of the polysaccharidesshowed that the former was built up predominantly of repeating-oligosaccharideunits of 3-O-rß-cellobiosyl-D-glucose and 3-O-rß-cellotriosyl-D-glucosein a molar ratio of 2.6 : 1.0, and the latter was of repeating-oligosaccharideunits of -D-xylosyl-(16)-rß-D-glucosyl-(14)-[-D-xylosyl-(16)]-rß-D-glucosyl-(14)-D-glucose,-D-xylosyl-(16)-rß-D-glucosyl-(14)-D-glucose and cellobiose. ¹ Present address: Department of Botany, Iowa State University,Ames, Iowa 50011, U.S.A. (Received August 29, 1981; Accepted January 12, 1982)     

Pyrone derivatives: effective inhibitors of photosynthetic electron flow system

The inhibitory effects of the pyrone derivatives, 6-()-decenyl-2,3-dimethyl--pyrone(DDP) and 6-farnesyl-2,3-dimethyI--pyrone (FDP), on the photosyntheticelectron flow system was investigated using the blue-green algaAnabaena variabilis and the green alga Chlorella pyrenoidosa. Both reagents inhibited photosynthesis in intact cells; 50%inhibition occurred at 2.7 x 10^–5 M with DDP and at 4.3 lO^–6 M with FDP in Anabaena photosynthesis. The reagentssuppressed the photosystem II reaction [water to 2,6-dichlorophenolindophenol (DCIP)] of Anabaena membrane fragments, but werefar less inhibitory on the photo-system I reaction (DCIPH₂ tomethyl viologen). The kinetics of the fluorescence inductionindicated that the reagents do not block Q-reduction, but dosuppress the oxidation of reduced Q indirectly. Oxygen evolutionunder repetitive flashes at a low repetition rate (5 Hz) wasinsensitive to the reagents even at concentrations which inducedmore than 50% inhibition. These results are evidence that DDPand FDP inhibit the plastoquinone reaction by slowing down itsturnover rate. The advantages of pyrone derivatives are that they are inactivein the oxidation-reduction reaction and do not quench the fluorescenceof chlorophyll in vivo. (Received April 14, 1980; )