Phycomyces: a new gene for a flavoprotein with covalently linked cofactor

Summary We have identified a new locus in Phycomyces blakesleeanus that codes for a flavoprotein designated previously as FP₂. According to results from a sexual cross, this locus, flp, maps near the gene madC, a marker for abnormal phototropic sensitivity. The recombination frequency between flp and madC is about 10%. Further, the flp marker is unlinked with the carotene locus carA. Because the flavoprotein FP₂ is absent in certain photoreceptor mutants (Pollock et al. 1985 a), it had been proposed as a candidate for the blue-light photoreceptor. We have found, however, that some strains lacking this protein retain normal phototropism. Therefore, the flavoprotein FP₂ is probably not involved in photoreception.     

The role of mutants in the search for the photoreceptor for phototropism in higher plants

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.     

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affected in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent components of the photoreceptor complex responsible for phototropism in Phycomyces.This work was supported in part by an equipment grant to JAP from the Syracuse University Senate Research Committee, research grants to EDL from the National Science Foundation (PCM-8003915 and DMB-8316458), and a fellowship to EDL from the Alfred P. Sloan Foundation.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Specific inhibition of phototropism in corn seedlings

Geotropism was used as a control for the specificity of potential inhibitors of phototropism by the coleoptiles of corn (Zea mays) seedlings. The compounds tested fall into three categories showing: (a) no inhibition of either phototropism or geotropism (KCl); (b) nonspecific inhibition of both phototropism and geotropism (KCN); and (c) specific inhibition of phototropism (KI, NaN₃, and phenylacetic acid). Simultaneous irradiation of coleoptiles with phototropically inert light in addition to the phototropically active blue light also results in an inhibition of phototropism. Since azide, iodide, and phenylacetic acid are known to interact with flavins while a simultaneous irradiation with a phototropically inert light may depopulate the first triplet state of flavins, these data support the hypothesis that the photoreceptor pigment for phototropism in corn is a flavin.     

Regulation of formation of covalently bound flavins in liver and cerebrum by thyroid hormones.

The effects of thyroxine (T₄) and triiodothyronine (T₃) treatment upon the formation of [2-¹⁴C]flavins bound covalently to tissue proteins in liver and cerebrum were measured 1 h after a subcutaneous injection of [2-¹⁴C]riboflavin in male rats of different ages. In livers of rats of ages 2, 3, and 12 months, T₄ (100 μg/100 g body wt) and T₃ (25 μg/100 g body wt) in daily intraperitoneal doses for 7 days each increased incorporation into covalently bound flavins 50% above that in saline-treated controls. In newborn rats, T₄ in doses of 10 μg/rat for 7 days increased incorporation similarly to that in adults. In adult rats doses of T₃ from 2.5 to 25 μg/100 g body wt were nearly as effective as larger doses of T₃ and T₄ in increasing the formation of covalently bound flavins in liver. In cerebra of newborn rats, T₄ was ineffective in increasing the formation of covalently bound flavins. However, in cerebra of rats of ages 2, 3, and 12 months, both T₃ and T₄ consistently increased the formation of covalently bound flavins. Doses of T₃ from 2.5 to 25 μg/100 g body wt produced significant increases. These findings are of interest in view of our previous demonstration that the formation of flavin adenine dinucleotide, the major tissue flavin, is not increased in rat brain even by massive doses of thyroid hormones. The present results indicate that the formation of the fraction of flavins bound covalently to tissue proteins differs from the usual pattern of brain metabolism of adult rats in being subject to control by thyroid hormones.     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants showed that CRY1-mediated blue-light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It was concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.     

Long-chain saturated fatty acids can be α-oxidised by a purified enzyme (Mr 240 000) in cucumber (Cucumis sativus)

An enzyme (M_r 240 000) with high fatty acid α-oxidation activity has been purified from the fruit of cucumber (Cucumis sativus). The specific α-oxidation activity in the purified fraction was 370 nmol/min per mg protein determined as liberation of ¹⁴CO₂ from [1-¹⁴C]palmitic acid. α-Oxidation activity was observed both in the 12 000×g pellet and 150 000×g pellet by differential fractionation of cucumber homogenate. The enzyme was purified about 220-fold to near homogeneity from a 12 000×g fraction by solubilisation with Triton X-100R, ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatographies and Superose 12 gel filtration. The molecular mass of the native enzyme was 240 000, and the major subunit molecular mass of 40 000 indicated an oligomeric structure.     

On insertion of pigment-associated polypeptides during membrane biogenesis in Rhodopseudomonas capsulata

Early stages in the formation of membranes and photosynthetic units were studied under growth-limiting phototrophic and chemotrophic conditions in cells of Rhodopseudomonas capsulata. The incorporation of polypeptides, forming bacteriochlorophyll-carotinoid-protein complexes in the membrane, was followed by use of pulse-labeling and immunoprecipitation techniques. The newly synthesized polypeptides were inserted into two distinct membrane fractions at both different rates and proportions. The two membrane fractions differed in sedimentation behavior, absorption spectra and activities of the respiratory chain. The individual pigment-associated proteins did not exhibit precursor-product relationship between the two membrane fractions. The data suggest that newly synthesized polypeptides were integrated both into cytoplasmic and pre-existing intracytoplasmic membranes, where the proteins and pigments were assembled to form reaction centers and light-harvesting pigment-protein complexes.Abbreviations Bchl bacteriochlorophyll - cpm counts per minute - M _r relative molecular mass - P 100 pellet of 100,000xg, 60 min - P300 pellet of 300,000xg, 90 min - pO₂ oxygen partial pressure - R Rhodopseudomonas - dodecyl sulfate sodium dodecyl sulfate. International standard units - Bq Becquerel (s^-1) - Pa Pascal (N/m²; 1 Torr=133,3 Pa)     

In-vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus

Several types of membrane-localized flavin binding sites were investigated in sporangiophores (spph) and mycelia of Phycomyces blakesleeanus. In-vitro binding of riboflavin, riboflavin-5′-phosphate, and flavin-adenine-dinucleotide was demonstrated with unfractionated membrane preparations by means of competition of [¹⁴C]riboflavin binding. Saturation of binding was only obtained with the highly water-soluble riboflavin-5′-phosphate, but by extrapolation it was shown that riboflavin showed the highest affinity towards the binding sites (K_D about 4·10^-6M). The number of binding sites was estimated to be 0.7 nmol g^-1 fresh-weight equivalent. Analysis of endogenous soluble flavin revealed that only riboflavin, riboflavin-5′-phosphate, and flavin-adenine-dinucleotide occurred in Phycomyces, and at a concentration of at least 1 nmol g^-1 fresh-weight equivalent in entire spph. Thus, the measured binding sites could reach saturation in-vivo. In the apical part of spph to which blue-light sensitivity is restricted, the amount of soluble flavin was three-fold higher. Exclusively in this zone, heat-labile riboflavin proteins were measured at a concentration of about 3 nmol g^-1 fresh-weight equivalent. The amount of covalently bound flavin was higher in spph tips than in intact spph (8 nmol and 3 nmol g^-1 fresh-weight equivalent, respectively). In either case, the concentrations of the flavin-membrane complexes were higher than the theoretical calculated concentration of (anisotropic) blue-light photoreceptor in Phycomyces (Bergman et al. 1969), and their involvement in blue-light photoreception is considered.     

In Vivo Generation of Flavoproteins with Modified Cofactors

To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Vacuolar localization of 1-sinapolglucose: l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus

The distribution of l-malate, sinapic acid esters and 1-sinapoylglucose: l-malate sinapoyltransferase (SMT) which catalyzes the synthesis of sinapoyl-l-malate were examined in preparations of protoplasts obtained from cotyledons of red radish (Raphanus sativus L. var. sativus). Vacuoles isolated from the protoplasts contained all of the SMT activity, all of the accumulated sinapic acid esters and about 50% of free l-malate present initially in the protoplasts. An esterase activity, acting on 1-sinapoyglucose, was found to be exclusively localized in the cytoplasm and a large proportion was found to be recoverable in a 100 000-g pellet obtained from protoplast lysates. The vacuoles were obtained after lysis of the protoplasts by osmotic shock and purification on a Ficoll gradient. The cytoplasmic contamination of vacuole preparations was found to be about 10%, as judged by enzymatic markers and microscopic inspection. No SMT activity was found in a 100 000-g pellet obtained from vacuole lysates. The results indicate that biosynthesis of sinapoyl-l-malate takes place within the central vacuoles of redradish cotyledons.Abbreviation SMT 1-sinapoylglucose: l-malate sinapol-transferase     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Induction,purification and characterisation of arabinases produced by Aspergillus niger

The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (-l-arabinofuranosidase A, -l-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83 000, 67 000, 43 000 Da and, pI 3.3, 3.5 and 3.0 for -l-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (K _m on p-nitrophenyl--l-arabinofuranoside 0.68 and 0.52 mM for -l-arabinofuranosidases A and B, resp.; K _m on sugar beet arabinan 0.24 and 3.7 g/l for -l-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commerical A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.Abbreviation PNA p-nitrophenyl--l-arabinofuranoside     

Lamivudine production via enantioselective deamination by thermostable Bacillus caldolyticus cytidine deaminase

To decrease the costs of producing the anti-HIV drug, lamivudine, an enzymatic conversion process was developed instead of the traditional chemical method. Thermostable cytidine deaminase was over-produced by cloning the cdd gene into E. coli JF611/pCJH53 from Bacillus caldolyticus. The purified cytidine deaminase was recovered from the lysate of the recombinant E. coli JF611/pCJH53 by removing heat-denatured proteins and eluting sequential chromatography. When the enzyme was used to deaminate (–)--l-(2R, 5S)- and (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine, about 68% of the (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine was deaminated into the corresponding (+)-thiauridine maximally.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Stigma proteins of the two loci self-incompatible grass Phalaris coerulescens

Summary Protein extracts from four self-incompatible genotypes of Phalaris coerulescens were subjected to analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultra-thin isoelectric focusing. A comparison between stigma, root and leaf extracts showed that there was no significant difference in electrophoretic or isoelectric focusing patterns between the genotypes for both root and leaf proteins. However, stigma protein patterns did vary between genotypes especially within the molecular weight region of 43 000–97 000 and within the pI range 5–7. The stigma-specific changes strongly suggest a link between the self-incompatible genotype and these stigma proteins. However, because there are two loci involved, it is not yet possible to precisely assign particular proteins to each S- or Z-allele.