Incorporation of some possible radioactive intermediates into chlorogenic acid in sliced sweet potato tissue

t-Cinnamic acid-2-¹⁴C, p-coumaric acid-2-¹⁴C and caffeic acid-2-¹⁴Cwere administered to discs of sweet potato roots and incorporationof each radioactive compound into chlorogenic acid was compared.The data suggest that chlorogenic acid is synthesized througheither or both of two major pathways, phenylalanine t-cinnamate t-cinnamoyl derivative p-coumaroyl derivative chlorogenicacid and phenylalanine t-cinnamate p-coumarate p-coumaroylderivative chlorogenic acid. ¹Part 75 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address : Department of Biology, Tokyo MetropolitanUniversity, Setagaya-ku, Tokyo. (Received December 23, 1968; )     

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; )     

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)     

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)     

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)     

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)     

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; )     

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)     

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; )     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

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     

Methionine as a Dominant Precursor of Phytosiderophores in Graminaceae Plants

Nine ¹⁴C-labeled amino acids and ¹⁴C-acetic acid from root tipsof Fe-deficient Graminaceae plants (Hordeum vulgare, Oryzaesativa and Avena saliva) were surveyed to determine the precursoramino acid of phytosiderophores. The dominant precursor wasmethionine, which was incorporated into avenic acid deoxymugineicacid mugineic acid epihydroxymugineic acid and/or hydroxymugineicacid in this order. Methionine sulfoxide or methionine sulfonemay be important intermediates in going from methionine to avenicacid. (Received May 6, 1987; Accepted June 12, 1987)     

Localization of the carbon in caffeine biosynthesized from N-methyl carbon of {gamma}-glutamylmethylamide in tea plants

1. Localization of carbon in caffeine molecule biosynthesizedfrom the N-methyl carbon of -glutamylmethylamide in tea plantswas observed. ¹⁴C-Caffeine produced from ¹⁴C--glutamylmethylamidewas isolated and degraded. Approximately 26–55% of the¹⁴C was observed in the three methyl carbons in caffeine, withonly 2–3% at the C-2 carbon, 3–7% at the C-8 carbonposition. The amount of ¹⁴C at the C-4, C-5 and C-6 positionswas calculated from the results obtained. 2. The role of the N-methyl carbon of -glutamylmethylamide inthe formation of RNA in tea plants was examined. Incorporationof the N-methyl-¹⁴C of ¹⁴C--glutamylmethylamide into AMP andGMP in RNA was found. These facts indicate that in tea plants, -glutamylmethylamideis metabolized and most of its N-methyl carbon is utilized asa precursor for caffeine formation and little, if any, as aprecursor for nucleic acid formation. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received February 2, 1972; )     

Effects of l, N6-ethenoadenylates on the regulation of photosynthetic activities by adenylates; A study of the nucleotide binding sites on chloroplast coupling factor 1

Effects of l, N⁶-ethenoadenylates (e-adenylates) were testedon phosphorylation, and electron transport under phosphorylation,arsenylation and quasi-arsenylation (stimulation of electrontransport in the presence of ATP, AMP and arsenate) conditionsin isolated spinach chloroplasts. -ATP as well as ATP partially inhibited ferricyanide reductionthrough binding to the chloroplast coupling factor 1 with anapparent dissociation constant (KD^app) of around 5µM,which was remarkably larger than that for ATP (ca. 2µM).e-ATP at below 500 µM had no effect on phosphorylationbut inhibited quasi-arsenylation in competition with ATP withan apparent inhibition constant (K₁^app) of around 60 µM. -ADP as well as ADP partially inhibited ferricyanide reductionwith a KD^app value close to that for -ATP. -ADP was phosphorylated(the apparent Michaelis constant, K_m^app=80µM) accompanyingstimulation of ferricyanide reduction to the magnitude predicted(P/_e=l). -ADP-arsenylation was also detected by stimulationof ferricyanide reduction. -AMP alone caused little inhibition of ferricyanide reductionas AMP, but competitively depressed the electron transport inhibitionby ADP and ATP with a K₁^app value of around 200 µM. -AMPwas not effective for ADP phosphorylation but inhibited stimulationdue to quasi-arsenylation coupling in competition with AMP K₁^app=150µM Among the possible combinations of adenylates and -adenylatesfor quasiarsenylation, only [ATP+AMP] could couple with theenergy transduction mechanism. Based on the specificity of binding sites to adenylates and-adenylates, an attempt was made to distinguish at least four(two pairs) kinds of binding sites (at least six sites in toto)on the chloroplast coupling factor 1 for photosynthetic energytransduction. When one pair of sites is occupied by the designatedadenylates or -adenylates (allosteric effectors), the couplingfactor is thought to be in a conformation for coupling withthe energy transduction mechanism in the presence of phosphateor arsenate. ¹Presented to the 1st Symposium of Japan Bioenergetics Group,December 19, 1975, Osaka. (Received February 17, 1976; )     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Long chain fatty acid synthesis in developing castor bean seeds II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system

Particles spun down at 10,000 ? g from developing castor beanseeds were capable of synthesizing LFAs from sucrose, a physiologicalprecursor transferred from leaves as a photosynthetic product.Tracer experiments, in combination with inhibitor effects, intermediatedilutions and cofactor requirements, indicated the operationof the following path: sucroseUDPGG-1-PG-6-PGAPpyruvateacetyl-CoAmalonyl-CoALFA.The whole path appears to be associated with 10,000 ? g particles,because repeated washings were unsuccessful in dissociatinga partial path from the particles, depsite of disorganizingthe structure of the particles. Based on the occurrence of freehexose(s) and the utilization of UDPG similar to that of sucroseor G-1-P in this reaction, it is probable that hexose phosphateis formed from sucrose via UDPG and fructose, although the conversionof sucrose to hexose phosphates via glucose and furctose isnot ruled out. Inhibitor experiments showed that ATP is self-supportingover the whole path, the ATP formed in the glycolytic path beingconsumed in a acetyl-CoA carboxylation step. Since oxidizedpyridine nucleotides are as available as reduced ones for LFAsynthesis, they seem to shuttle between the reduction in theconversion of sucrose to acetyl-CoA and the oxidation in LFAsynthesis from acetyl-CoA. From the pattern of the LFAs synthesized,NAD⁺ is available for the synthesis of saturated LFAs (18: 0,16: 0). whereas NADP⁺ is available for that of unsaturated LFAs(18: 1, 16: 1). (Received July 23, 1973; )     

Characterization of the Hydrolytic Activity of Avocado Cellulase

The cellulase produced by ripening avocado fruits (Persea americanaMill cv. Fuerte) was isolated and purified using chromatofocusing(pH 7–4) and gel filtration on a Bio- Gel P-100 column.Characteristics of the cellulase were assessed by using, assubstrates, a range of polysaccharides containing various sugarresidues and varying types of linkages between the residues.Only those substrates containing (14)-ß-glucosyl linkageswere hydrolyzed by the purified enzyme. Two polysaccharidesthat were extensively hydrolyzed by the cellulase were carboxymethylcelluloseand (13),(14)-ß-D-glucans such as from Avena endospermcell walls. Characterization of the activity in the degradationof the mixed linked glucan of Avena and cellodextrins indicatedthat the enzyme has a limit recognition-hydrolytic site of four(l4)-ß linked glucose residues. It was also foundthat the enzyme could cleave only (14)-ß-linkagesthat were adjacent to other (l4)-ß-D-glucosyl linkages.Activity of the cellulase against isolated avocado fruit cellwalls indicated that the purified enzyme was incapable of appreciablysolubilizing the cellulosic components of these walls. ¹Supported in part by National Science Foundation Research GrantPCM 7818588. ²USDA-ARS, Dairy Forage Research Center, University of Wisconsin,Madison, WI 53706. ³Department of Vegetable Crops, University of California, Davis,CA 95616. (Received September 14, 1985; Accepted February 12, 1986)     

AGMATINE AND N-CARBAMYLPUTRESCINE AS INTERMEDIATES IN THE FORMATION OF NICOTINE BY TOBACCO PLANTS

Agmatine-G-³H and N-carbamylputrescine-l,4-¹⁴C were effectivelyincorporated into the nicotine of tobacco plants. This resultmay indicate a route that the pyrrolidine ring of nicotine isformed from putrescine by the following pathway: arginineagmatineN-carbamylputrescineputrescinepyrrolidinering. (Received February 7, 1966; )