Hydrolysis of riboflavin in plants

The enzymic hydrolysis of riboflavin to lumichrome and ribitol by extracts of Crinum longifolium bulbs has been demonstrated. The enzyme was purified 48-fold by ZnSO4 treatment and ethanol fractionation, and concentrated by using Sephadex G-25. After establishing the stoichiometry of the reaction, the general properties of the purified enzyme were studied. The enzyme showed maximal activity at pH 7·5, and it had a requirement for reduced glutathione which could be replaced by cysteine or ascorbic acid. Mg2+ and Li+ activated the enzyme. The reaction was highly specific to riboflavin and was competitively inhibited by riboflavin 5′-phosphate.

Biosynthesis of valine and isoleucine in plants I. Formation of α-acetolactate in Phaseolus radiatus

1. 1. The presence of an enzyme system in plants catalyzing the formation of α-acetolactate from pyruvate has been demonstrated; the system in green gram (Phaseolus radiatus) has been partially purified and its characteristics have been studied.2. Free acetaldehyde is formed as a product of the reaction and so the reaction is mainly diverted towards the formation of acetoin. 3. The system requires thiamine pyrophosphate and a divalent metal ion (Mn2+ or Mg2+) for maximum activity. The optimum pH is around 6.0 and the optimum temperature is 60°. 4. The system is very labile in absence of pyruvate, Mn2+ and DPT. 5. The Km values for pyruvate, Mn2+, Mg2+ and DPT are 3·10−2 M. 5·10−5 M, 2·10−5 M, and e·10−6 M respectively. The activation energy is 3540 cal/mole. 6. The enzyme is strongly inhibited by p-chloromercuribenzoate and the inhibition can be reversed partially by 2-mercaptoethanol, BAL or cysteine. Heavy metals, such as Hg2+ and Ag+, are inhibitory but l-valine does not inhibit the reaction.

Studies on the enzyme hydrolysing flavin mononucleotide in plants: I. Partial purification and properties of the enzyme from Phaseolus radiatus

The partial purification of the enzyme hydrolysing FMN from extracts of greengram seeds (Phaseolus radiatus) is described. The procedures, which entailed precipitation of inert material by manganous sulfate and protamine sulfate treatment, fractional precipitation with alcohol and chromatography on CM-cellulose, afforded preparations whose specific activity was 200 times that of the initial crude extract. The preparation was comparatively specific for FMN. It also hydrolysed, to a much smaller extent, β-glycerophosphate, p-nitrophenyl phosphate and 5′-nucleotides. The differential effects of ions on the FMN and β-glycerophosphate hydrolysing activities are discussed.

Studies on the enzyme hydrolysing flavin mononucleotide in plants. I. Partial purification and properties of the enzyme from Phaseolus radiatus

The partial purification of the enzyme hydrolysing FMN from extracts of greengram seeds (Phaseolus radiatus) is described. The procedures, which entailed precipitation of inert material by manganous sulfate and protamine sulfate treatment, fractional precipitation with alcohol and chromatography on CM-cellulose, afforded preparations whose specific activity was 200 times that of the initial crude extract. The preparation was comparatively specific for FMN. It also hydrolysed, to a much smaller extent, β-glycerophosphate, p-nitrophenyl phosphate and 5′-nucleotides. The differential effects of ions on the FMN and β-glycerophosphate hydrolysing activities are discussed.

Studies on the enzyme hydrolysing flavin mononucleotide in plants: II. Phosphotransferase activity of the partially purified enzyme from Phaseolus radiatus

The preparation of the enzyme hydrolysing FMN whose partial purification from green-gram extracts is described in the preceding paper, has been shown to possess phosphotransferase activity. The enzyme could transfer the phosphate group cleaved from FMN to acceptors like thiamine, pyridoxal, pyridoxamine and nucleosides resulting in the formation of their corresponding phosphate esters and nucleotides. The properties of the enzyme hydrolysing FMN and the phosphotransferase activity of the preparation are compared.

Tryptophan synthetase in plants

Infiltration experiments with the intact seeds of Bengal gram (Cicer arietinum) indicated that indole and serine are the immediate precursors of tryptophan in this legume. The enzyme, tryptophan synthetase, has been demonstrated in cell-free extracts of the resting seeds. The optimum pH of the reaction was 5.5, and the Km value for indole at a constant serine concentration of 10−4M was 0.57 × 10−4M. There was a specific requirement for pyridoxal phosphate. Heavy-metal ions were inhibitory.

Lihaluujauhon soveltuvuus sokerijuurikkaan lannoitteeksi

Lihaluujauho muodostaa maatilojen myytävien kasvi- ja eläinperäisten tuotteiden jälkeen tärkeimmän agroekosysteemeistä poispäin suuntautuvan ravinnevirran. Se sisältää runsaasti pääkasvinravinteita typpeä, fosforia ja kalsiumia (N ~8%, P ~5%, Ca yleensä ~10-15% luuaineksen määrästä riippuen), sekä kaliumia n.1% tai alle. Lihaluujauho on todettu tehokkaaksi lannoitteeksi useilla viljelykasveilla ja sen käyttö on sallittu myös luomuviljelyssä EU-alueella. Lihaluujauhoon ja erityisesti sen rehukäyttöön liittyvistä riskeistä merkittävin on TSE-tautien riski (naudan BSE-, lampaiden ja vuohien scrapie-, sekä ihmisen vCJD-taudit). Rehukäyttöä on monissa maissa rajoitettu 1980-luvulla puhjenneen BSE-kriisin myötä. BSE-taudin leviäminen yhdistettiin tilanteeseen, jossa nautaperäistä lihaluujauhoa käytettiin nautaeläinten rehun ainesosana. Myös lihaluujauhon käytössä turkiseläinrehuna saattaa piillä BSE:n tai muun TSE-taudin riski. Oikein käsitellyn lihaluujauhon lannoitekäyttöön ei kuitenkaan näytä tarkastelemieni tutkimusten perusteella sisältyvän huomattavaa TSEriskiä, jos huolehditaan asianmukaisista varotoimista ja menettelyistä sekä tuotteen valmistusprosessissa, että käytettäessä lannoitetta. Lihaluujauhon lannoitekäytön lisääminen edistäisi ruokajärjestelmämme ravinnekierron sulkemista etenkin fosforin osalta. Lihaluujauho on uusiutuva luonnonvara, jonka lannoitekäytöllä voitaisiin korvata huomattava osa lannoiteaineena kulutettavista fosforipitoisista kiviaineista. Sokerijuurikkaan lannoituskokeissa Varsinais-Suomen Kaarinassa vuosina 2008 ja 2009 lihaluujauhokäsittelyt eivät menestyneet aivan yhtä hyvin satotasovertailussa kuin kontrollikäsittelyiden NPK-väkilannoitteet, mutta laatuominaisuuksiltaan (sokeripitoisuus, amino-N, K, ja Na-pitoisuudet) joiltakin osin kontrollikäsittelyjä paremmin. Kokeissa käytetyt lajikkeet olivat ’Jesper’ vuonna 2008 ja ’Lincoln’ vuonna 2009. Käytetty lihaluujauholannoite oli Honkajoki Oy:n Viljo Yleislannoite 8-4-3, joka sisälsi noin 10% kaliumsulfaatin ja kasviperäisten sivutuotteiden seosta. Viljo-lannoitetta käytettiin sekä yksistään, että yhdistettynä 10-25%:iin väkilannoitetta. Vuoden 2009 Viljo-koejäseniin vielä lisättiin kaliumsulfaattilannoitetta (42% K, 18% S), jotta päästiin annetun kaliumin määrässä päästiin lannoitussuosituksen (60 kg K/ha) tasolle. Pelkkä Viljo-lannoite tuotti merkitsevästi alhaisemmat sadot kuin kontrollikäsittelyt molempina vuosina. Kuitenkin kun Viljolannoitteen ohella käytettiin väkilannoitetta (10-25% kasvin typentarpeesta) päästiin varsin lähelle kontrollikäsittelyiden satotasoja. Myös pelkän LLJ-lannoitteen tuottamat satotasot olivat kuitenkin selvästi paremmat kuin Suomen keskimääräiset juurikassadot. Viljo-käsittelyillä oli selvästi positiivinen vaikutus laatutekijöihin amino-N, K ja Na vuonna 2008, mutta vuonna 2009 näiden pitoisuudet jäivät kontrollikäsittelyjen tasolle. Viljo-käsittelyiden sokeripitoisuudet olivat vuonna 2008 kontrollikäsittelyn luokkaa ja Viljo77%+NK1:n osalta kontrollia merkitsevästi paremmat. Vuoden 2009 sokeripitoisuudet olivat kaikilla koejäsenillä erinomaiset, ja käsittelyiden välillä ei ilmennyt merkitseviä eroja. Kokeiden perusteella kaliumsulfaatilla täydennetty lihaluujauho on hyvin toimiva lannoite sokerijuurikkaalla Suomen olosuhteissa, etenkin yhdistettynä väkilannoitteeseen.

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Determination of distances of sugar protons from Mn2+ in concanavalin A

1H NMR spin-lattice relaxation time (T1) measurements have been carried out with various sugars, viz. methyl alpha-D-glucopyranoside (alpha-MeGluP), methyl beta-D-lucopyranoside (beta-MeGluP), methyl alpha--annopyranoside (alpha-MeManP), maltose (4-O-alpha-D-glucopyranosyl--glucose), nigerose (3-O-alpha-D-glucopyranosyl-D-glucose), p-nitrophenyl alpha-maltoside (PNP-alpha-maltoside) and p-nitrophenyl beta-maltoside (PNP-beta-maltoside) to determine the distances of sugar protons from Mn2+ in concanavalin A (Con A). With a rotational correlation time of 1.58 x 10(-10) s determined, distances were calculated using Solomon-Bloembergen equation. The data obtained indicated differences in disposition of different groups in the binding site of Con A. An average value of about 10 A was obtained for the distances of sugar protons from Mn2+ in Con A. In the case of mono and disaccharides, the non-reducing end sugar unit was found to be closer to Mn2+ than the reducing end one.