The relationship between different spectral forms of the protochlorophyllide oxidoreductase complex and the structural organisation of prolamellar bodies isolated from <Emphasis Type="Italic">Zea mays</Emphasis>

Incubation of prolamellar bodies (PLB) in high-salt media leads to changes in PLB structure and properties of their protochlorophyllide oxidoreductase–protochlorophyllide (POR–PChlide) complex. The paracrystalline organisation typical of PLB is disrupted and NADPH dissociates from photoconvertible POR–PChlide, with absorption maxima at 640 and 650 nm (POR–PChlide _640/650 ), and a non-photoconvertible form, with absorption maxima at 635 nm (POR–PChlide ₆₃₅ ), is formed. These effects are strongly dependent on the valence of the cation of the perturbing salt, indicating that they involve surface double layers effects. They are also influenced by the nature of the anion and by high concentrations of non-electrolytes, suggesting the involvement of surface hydration effects. The structural changes are largely, if not entirely, independent of the presence of excess NADPH. Changes to the POR–PChlide complex, however, are strongly inhibited by excess NADPH suggesting that the two sets of changes may not be causally linked. As long as the disruption is not too great, the structural changes seen on incubation of PLB in high salt media lacking excess NADPH are reversed on removal of the high salt perturbation. This reversal is independent of the presence or absence of added NADPH. Reformation of photoconvertible POR–PChlide, however, requires the presence of NADPH. The reformation of paracrystalline PLB in the absence of NADPH strongly indicates that preservation of PLB structure, in isolated PLB preparations at least, is independent of the presence or absence of POR–PChlide ₆₅₀ .     

Far-red induced changes of the protochlorophyllide components in wheat leaves

Biosynthesis of chlorophyll is partly controlled by the phytochrome system. In order to study the effects of an activated phytochrome system on the protochlorophyllide (PChlide) biosynthesis without accompanying phototransformation to chlorophyll, wheat seedlings (Triticum aestivum L. cv. Starke II Weibull) were irradiated with long wavelength far-red light of low intensity. Absorption spectra were measured in vivo after different times in the far-red light or in darkness. The relationship between the different PChlide forms, the absorbance ratio _{650nm636 nm} changed with age in darkness, and the change was more pronounced when the leaves were grown in far-red light. Absorption spectra of dark-grown leaves always showed a maximum in the red region at 650 nm. For leaves grown in far-red light the absorption at 636 nm was high, with a maximum at the 5 day stage where it exceeded the absorption at 650 nm. At the same time there was a maximum in the total amount of PChlide accumulated in the leaves, about 30% more than in leaves grown in darkness. But the amount of the directly phototransformable PChlide, mainly PChlide_650–657, was not increased. The amount of PChlide_628–632, or more probably the amount of (PChlide_628–632, + PChlide _636–657) was thus higher in young wheat leaves grown in far-red light than in those grown in darkness. After the 5 day stage the absorption at 636 nm relative to 650 nm decreased with age, and at the 8 day stage the spectra were almost the same in both types of leaves. Low temperature fluorescence spectra of the leaves also showed a change in the ratio between the different PChlide forms. The height of the fluorescence peak at 632 nm relative to the peak at 657 nm was higher in leaves grown in far-red light than in dark-grown leaves. – After exposure of the leaves to a light flash, the half time for the Shibata shift was measured. It increased with age both for leaves grown in darkness and in far-red light; but in older leaves grown in far-red light (7–8 days) the half time was slightly longer than in dark-grown leaves. – The chlorophyll accumulation in white light as well as the leaf unrolling were faster for leaves pre-irradiated with far-red light. The total length of the seedlings was equal or somewhat shorter in far-red light, but the length of the coleoptile was markedly reduced from 8.1 ± 0.1 cm for dark-grown seedlings to 5.2 ± 0.1 cm for seedlings grown in far-red light.     

Polyvalent GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (T <Subscript>α</Subscript>) as the most potent recognition factors involved in <Emphasis Type="Italic">Maclura pomifera</Emphasis> agglutinin–glycan interactions

Summary The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Gal13GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin–glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAc1Ser/Thr (Tn) and Gal13GalNAc1Ser/Thr (T) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 × 10⁵, 4.6 × 10⁴ and 3.9 × 10⁴ more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a⁺) related disaccharide (GalNAc14Gal) and P^k/P₁ active disaccharide (Gal14Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA–glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T (M.W. > 4.0 × 10⁴) >> Tn-containing glycopeptides (M.W. < 3.0 × 10³) > monomeric T/Tn and P (GalNAc13Gal) > GalNAc > Gal >> Man, LAra, DFuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Purification and initial characterization of phycobiliproteins from the endophytic cyanobacterium of Azolla

The endophytic cyanobacterium, Anabaena azollae, isolated from laboratory cultures of Azolla caroliniana Willd., contains three spectroscopically distinct biliproteins. About 70% of the biliprotein is c-phycocyanin (_max 610 nm) and 13% is allophycocyanin (_max 647 nm, shoulder 620 nm). A third pigment corresponds to phycoerythrocyanin (_max 570 nm, shoulder 590 nm). In very dilute solutions of allophycocyanin, at constant pH and buffer strength, the 647 nm maximum disappears and a single _max occurs at 615–620 nm. The 647 nm absorption maximum reappears upon concentrating the dilute solution. Very dilute solutions of phycoerythrocyanin exhibit a broad peak between 570 and 590 nm. Absorption spectra of c-phycocyanin are not significantly altered upon dilution. Fluorescence emission maxima of phycoerythrocyanin, c-phycocyanin, and allophycocyanin occur at 630 nm, 643 nm and 660 nm respectively, using 540 nm excitation. Two subunits, of molecular weight 16,500 () and 20,600 (), are seen in c-phycocyanin upon dissociation with SDS. Dissociation of allophycocyanin and phycoerythrocyanin with SDS yields one sizeclass of subunits, with a molecular weight of about 17,500 for allophycocyanin and 18,000 for phycoerythrocyanin.Contribution No. 684 Offprint requests to: G. A. Peters     

Phytochrome modulation of ATPase activity in a membrane fraction from Phaseolus

Membrane-bound phytochrome and ATPase (ATP phosphohydrolase EC 3.6.1.3.) activity extracted from hypocotyl hooks of etiolated Phaseolus aureus Roxb. were both separated from solute proteins by gel filtration on Sepharose C1-2B. The amount of phytochrome detected in the membrane fraction was very small and was not significantly increased by red irradiation (in vivo or in vitro). Membrane-bound ATPase activity was modulated in vitro by the phytochrome in the membrane fraction, being lower after red light than after far-red light. This effect was potentiated by a preliminary light reaction which occurred only in vivo and, in continuous red light, required 60 to 90 s at 25°C. Thus a two minute, in vivo, red irradiation reduced membrane-bound ATPase activity to about half that of the etiolated state. Subsequently bound-ATPase activity was determined by the form of phytochrome (P_r or P_fr) irrespective of whether established in vivo or in vitro. These results indicate that binding or release (of enzyme, cofactors or inhibitors) is not involved in phytochrome modulation of enzyme activity in the membrane fraction.Abbreviations R red light - F far red light - P_r inactive form of phytochrome (_max=660 nm) - P_fr active form of phytochrome (_max=730 nm) - MOPS N-morpholino-3-propansulphonic acid     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Fumonisin production by <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from freshly harvested corn in Iran

Fifty-one strains of Fusarium verticillioides and F. proliferatum isolated from corn collected from four different geographic areas in Iran, namely Fars, Khuzestan, Kermanshah and Mazandaran (an endemic oesophageal cancer (OC) area) were evaluated for their ability to produce fumonisins B₁ (FB₁), B₂ (FB₂) and B₃ (FB₃) in corn culture. Fumonisin levels were determined by high-performance liquid chromatography. All tested strains of F. verticillioides and F. proliferatumproduced fumonisins within a wide range of concentrations, 197–9661 g/g, 18–1974 g/g, and 21–1725 g/g for FB₁, FB₂, and FB₃, respectively. The highest mean concentrations of FB₁, FB₂, and FB₃ were 3897, 806 and 827 g/g, respectively. Overall, 61% of the F. verticillioides and F. proliferatum strains produced higher levels of FB₃ than FB₂. The mean ratios of FB₁:FB₂, FB₁:FB₃ and FB₁:total fumonisins were 8, 7 and 0.7 for F. verticillioides and 5.7, 10.7 and 0.7 for F. proliferatum, respectively. Significant differences in some of the meteorological data (rainfall, relative humidity and minimum temperature) from the four provinces were observed. Fumonisin levels produced by F. verticillioides strains isolated from Khuzestan province (tropical zone) were significantly (P < 0.01) higher than the other three provinces. This is the first report of the fumonisin-producing ability of F.verticillioides and F. proliferatum strains isolated from corn harvested from different geographic areas in Iran.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Histological examination of spruce needles from a long-term gas exchange experiment in pure and polluted air in the field

Summary At the end of a 4-year period of gas exchange measurements in a natural stand in the Lower Bavarian Forest, needles of an adult spruce [Picea abies (L.) Karst.] were harvested from two chambers, one with pure air and the other with ambient air. The needles were examined as to their histological properties in the stomatal apparatus and in the bundle sheath. In needles from the polluted air UV absorbance at 280 nm was decreased in the walls of the stomatal apparatus. Simultaneously, the deposition of compounds with an absorption maximum at 310 nm increased within the encrusted plate-like thickenings of the subsidiary cells. The contents of the lumina of hypodermal cells and of the bundle sheath exhibited a greater degree of autofluorescence in ambient-air material than in pure-air leaf organs. Differences between needles exposed to pure and polluted air are gradual. The damaged condition is rare in pure air, common in polluted air. The needles from outside the chambers occupied an intermediate position between pure-air and ambient-air needles. This fact is traced to an unnaturally high pollutant load in the liquid phase of the needle surfaces within the ambient-air chamber because in order to compensate pollutant losses within the system, SO₂ and O₃ were added even during periods of irrigation. The reduction of absorption capacity at 280 nm in the walls of the stomatal apparatus is attributed to destruction of lignin due to the high reactivity of the pollutants in the liquid phase on the damp needle surface. The importance of delignification with regard to hydroregulation is discussed.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Isolation and partial characterization of basic proteinases from stem bromelain

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosineserine) and position 20 (asparagine glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.02.01.02.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K _m 2.30 mM,k _cat 0.87 sec^–1 and F5 (K _m 2.42 mM,k _cat 0.68 sec^–1), and differed greatly from F9 (K _m 0.40 mM,k _cat 3.94 sec^–1).Dedicated to H. Tschesche, Bielefeld, Germany, on behalf of his 60th anniversary.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

Cryptomonad biliprotein: Phycocyanin-645 from a Chroomonas species

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution.Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm.The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below.Phycocyanin-645 is heterogenous in charge as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment components differing in mobility were found. We propose the term multiple pigment forms for these five phycocyanin-645 modifications.Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (₁, ₂), having molecular weights of 9200 and 10400, respectively, and one heavy chain (), having a molecular weight of 15500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is _{1 1}-_{2 1}.Abbreviations PC-645 phycocyanin-645 - C-PC C-phycocyanin - SDS sodium dodecyl sulphate - pI isoelectric point - mol. wt. molecular weight     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

Oxidation of ethylene by cotyledon extracts from Vicia faba L.

Improved rates of ethylene oxidation by cell-free preparations from cotyledons of Vicia faba L. have been obtained using cryogenic storage techniques and by developing a method for the hydrolysis of ethylene oxide. Gel permeation chromatography showed that a low-molecular-size fraction was required for activity; accordingly, the kinetics of ethylene oxidation in the presence of this fraction were studied. Reduced pyridine nucleotides could substitute for the low-molecular-size fraction. Activity under a nitrogen atmosphere was 60% lower than in air. The need for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen indicated that the enzyme might be a mixed-function oxidase. Using sufficient NADPH to approach saturation, the apparent Michaelis constant (K _m) for ethylene was 1.94±0.38 · 10^-8 M (aqueous phase), and when ethylene was saturating, the K _m for NADPH was 3.7 · 10^-5 M. Carbon monoxide was found to inhibit by competing with ethylene, and the inhibitor constant was 5.97 · 10^-7 M in solution. In the presence of excess ethylene and NADPH, activity was highest in phosphate-buffered medium pH 7.9. The bulk of the activity was found in a microsomal fraction.Abbreviations Epps N-2-hydroxyethylpiperazine-N-3-propane sulphinic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-porpanediol     

The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations

The rate of CO₂ fixation (F_c) and 680 nm chlorophyll fluorescence emission (F680) were measured simultaneously during induction of photosynthesis in Zea mays L. leaves under varying experimental conditions in order to assess the validity of fluorescence as an indicator of in vivo photosynthetic carbon assimilation. Z. mays leaves showed typical Kautsky fluorescence induction curves consisting of a fast rise in emission (O to P) followed by a slow quenching via a major transient (S-M) to a steady-state (T). After an initial lag, net CO₂ assimilation commenced at a point corresponding to the onset of the S-M transient on the F680 induction curve. Subsequently, F_c and F680 always arrived at a steady-state simultaneously. Decreasing the dark-adaption period increased the rate of induction of both parameters. Alteration of leaf temperature produced anti-parallel changes in induction characteristics of F_c and F680. Reducing the CO₂ level to below that required for saturation of photosynthesis also produced anti-parallel changes during induction, however, at CO₂ concentrations tenfold greater than the atmospheric level the rate of F680 quenching from P to T was appreciably reduced without a similar change in the induction of F_c. Removal of CO₂ at steady-state produced only a small increase in F680 and a correspondingly small decrease in F680 occurred when CO₂ was re-introduced. The complex relationship between chlorophyll fluorescence and carbon assimilation in vivo is discussed and the applicability of fluorescence as an indicator of carbon assimilation is considered.Abbreviations F_c rate of CO₂ fixation - F680 fluorescence emission at 680 nm     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Kinetic mechanism of NADP-malic enzyme from maize leaves

The kinetic mechanism of NADP-dependent malic enzyme purified from maize leaves was studied in the physiological direction. Product inhibition and substrate analogues studies with 3 aminopyridine dinucleotide phosphate and tartrate indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism. NADP is the leading substrate followed by l-malate and the products are released in the order of CO₂, pyruvate and NADPH. The enzyme also catalyzes a slow, magnesium-dependent decarboxylation of oxaloacetate and reduction of pyruvate and oxaloacetate in the presence of NADPH to produce l-lactate and l-malate, respectively.     

Adaptation of microbial activities to the environmental conditions in alpine soils

Summary The adaptation of soil microorganisms to different environmental conditions was investigated in the Austrian Central Alps (Hohe. Tauern). The floristic composition of the soil fungi at different sites was determined and the CO₂-release from soils taken from different altitudes was measured at different temperatures. The results showed a decreasing diversity of soil fungi with increasing altitude and a change in the dominating species at different altitudes and/or with vegetation patterns. The relative rates of CO₂-release from soils from different altitudes did not differ at different incubation temperatures. It was concluded that, among soil fungi the selection of species is a more effective mechanism for the adaption to changed environmental conditions than metabolic adaptations.This study was supported in part by the Österreichische MaB-Hochgebirgsprogramm Hohe TauernDedicated to Dr. K.F. Springer