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)     

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; )     

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; )     

IDENTITY OF {varepsilon}-CAROTENE AND {varepsilon}1-CAROTENE

-Carotene was isolated from previously known sources and itsspectral and adsorption properties compared with those of asimilar carotene recently observed in Cryptomonas ovata. Identitywas established. Similar comparisons with synthetic ₁-carotenepoint to the identity of the natural and synthetic compoundsand permit assignment of KARRER and EUGSTER's formulation for₁-carotene to the naturally occurring representative. ¹ Dedicated to Prof. H. TAMIYA on the occasion ot his 60th birthday. ²Contribution from the Scripps Institution of Oceanography,University of California, San Diego. (Received January 22, 1963; )     

Water-Relations Parameters of the Phloem. Determinations of Volumetric Elastic Modulus and Membrane Conductivity using an Applied Force Method and Shrinkage and Swelling of Tissues in Solutions at Differing Osmotic Pressure

Water-relations parameters were measured on sections of secondaryphloem from red oak (Quercus borealis michx. f.) and white ash(Fraxinus americana var. biltmoreana [Beadle] J. Wright) usinga linear displacement transducer. Changes in tissue thicknessin response to changes in the osmotic pressure of the bathingsolution were used to calculate the volumetric elastic modulusplus osmotic pressure (_v + ) of the tissue, and an applied forcemethod was used to estimate the time constant for water equilibration(T). The hydraulic conductivity of the cell membranes (L_p) wascalculated utilizing _v + and r values. The time-dependent behaviour of the tissue was much more complexthan originally expected. A correction for a time-dependentprocess that we call ‘drift’ was required to obtainnumbers for _v + . Furthermore, _v + was calculated on two assumptionsin order to relate changes in tissue dimensions to sieve elementparameters. In the first case, a lower limit for _v + of thesieve elements was determined by attributing all changes intissue dimensions to these cells. For red oak the average _v+ on this assumption is 72 bars. Assuming that all cell typeswere equally responsible for the changes in tissue dimensionsresulted in an _v + value of 192 bars for oak. If _v + and rare the same for all cells in the tissue, L_p for the sieve elementsof oak is 9.6 x 10^–8 cm s^–1 bar^–1. Exudationfrom the sieve elements of white ash during excision of thephloem led to artificially high values of _v + for that species. Quercus borealis michx. f., Fraxinus americana var, biltmoreana (Beadle) J. Wright, red oak, white ash, water relations, phloem, volumetric elastic modulus, membrane hydraulic conductivity     

The Derivation of the Cell Wall Elasticity Function from the Cell Turgor Potential

An equation is derived expressing average turgor pressure ofa leaf (_p) as a function of relative water content (RWC). Basedon this derivation, the relationships of the bulk elastic modulus(_v) and both RWC and _p, are formulated and discussed. The bulkelastic modulus (_v) becomes zero for _p = 0, that is at the turgorloss point for the leaf. At full water saturation the valueof ev is proportional to the water saturation turgor potential_p(max). The factor relating _P and _v (structure coefficient ,Burstrom, Uhrstr?m and Olausson, 1970) changes only very littlefor values of _p, which are not too close to zero. An exampleis given for the calculation from experimental data of the turgorpressure function, the structure coefficient function, and the_v function. Key words: Cell wall, Turgor pressure, Bulk elastic modulus     

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)     

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; )     

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; )     

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)     

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; )     

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)     

Part of the Lysyl Residues in Wheat {alpha}-Amylase is Methylated as N-{varepsilon}-Trimethyl Lysine

Amino acid analyses of wheat -amylase purified from germinatingseeds by affinity chromatography showed a high content of amodified lysyl residue. The modified residue was identifiedas N--trimethyl lysine. The presence of trimethyl lysine in-amylase is discussed in terms of isozymes. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received August 20, 1981; Accepted March 19, 1982)     

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; )     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

Osmotic Properties of Drought Stressed Periwinkle (Catharanthus roseus) Genotypes

The pressure-volume technique was employed to compare waterrelations and moisture stress-induced osmotic adjustment ofPeriwinkle (Catharanthus roseus) cv. Pink (PC), Oscillatus (REC)and White (WC). Leaf water potential (_w), osmotic potential(_s), turgor potential (_p), bulk modulus of elasticity (), boundwater (RWC_w) and leaf hydration (H), were estimated by exposingthe plants to a drying cycle during which well watered plantswere dehydrated to zero turgor, and then irrigated. Osmoticadjustment (_w ¹⁰⁰) was calculated by comparing _a at full hydration(_a ¹⁰⁰) in stressed plants after recovery, with _a ¹⁰⁰ in controlplants. Values of ₂¹⁰⁰ were 0.76, 0.33 and 0.11 MPa in cv. PC,REC and WC, respectively. Maintenance of _p at lower ₃ and relativeleaf water content (RWC) in prestressed PC was attributableto a higher alkaloid content and greater leaf cell wall elasticity.RWCW was plotted against _p to determine its contribution tohydration maintenance at lower _p. Genotype PC showed greaterRWC_w at lower _p compared with REC and WC. The present studyhas demonstrated that there are cultivar differences in alkaloidaccumulation and water relations in acclimated plants and thatthe relative ranking for drought resistance within periwinkleappeared to correspond with the changes in osmotic properties. Medicinal plant, drought resistance, alkaloids, periwinkle [Catharanthus roseus (L.) G. Don]     

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 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; )