Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations BrdU 5-bromo-2-deoxyuridine - DAPI 4-6-diamidino-2-phenylindole - DiOC₆a 3,3-dihexyloxacarbocyanine - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - ptDNA plastid DNA - pt-nucleoid plastid nucleoid - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by grant No. 2553 to M.F. and Nos. 04454019, 03304005 and 06262204 to T.K. from the Ministry of Education, Science and Culture of Japan, and by a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries of Japan.     

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity

Metabolism of 2-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO₂ assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.Abbreviations CA 2-carboxy-D-arabinitol - CA1P 2-carboxy-D-arabinitol 1-phosphate - CABP 2-carboxy-D-arabinitol-1,5-bisphosphate (transition state analog) - PFD photon flux density - P₁ inorganic phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate     

Heat modification of ribulose-1,5-bisphosphate carboxylase/oxygenase by temperature pretreatment of wheat (Triticum aestivum L.) seedlings

The effect of low-, ambient- and high-temperature pretreatments (48 h at 4° C, 20° C or 36° C) of wheat seedlings (spring wheat Triticum aestivum L., cv. Kolibri) on the solubility properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase; EC 4.1.1.39) was studied. The extractable protein moiety of heat-pretreated plants exhibited increased solubility in dilute buffer (50 mM k-phosphate, pH 6.8), compared with protein extracted from 4° C- or 20° C-plants. The salting-out characteristics for ammonium-sulfate precipitation confirmed this finding since a delayed precipitation of extractable protein from 36°C-plants was observed. Using polyacrylamide gel electrophoresis, the in-vivo temperature-induced differences in protein solubility could be traced back to a change in the solubility of RuBPCase. The RuBPCase was purified from wheat seedlings, and the purified enzyme also exhibited differential solubility. In order to evaluate this further, purified RuBPCase was subjected to probing for conformational properties. A decrease of fluorescence of the RuBPCase 1-anilino-8-naphtalene sulfonate complex revealed that the RuBPCase from 36° C-plants had a more hydrophilic protein surface. Titration of the sulfhydryl groups of native RuBP-Case with 5,5-dithiobis (2-nitrobenzoic acid) pointed to a reduced accessibility of the R-SH groups in the case of the 36° C-type of RuBPCase. The large subunit of RuBPCase from 4° C/20° C-plants tended to give rise to an artificial lower-molecular-weight polypeptide which could not be found in crude or purified RuBPCase from heat-pretreated wheat seedlings.Abbreviations ANS 1-anilino-8-naphtalene sulfonate - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bis-phosphate carboxylase/oxygenase - RuBP ribulose-1,5-bis-phosphate     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Inhibition of photosynthesis and respiration by batatasins

Effects of batatasins I, III and V, phenolic growth inhibitors occuring in dormant bulbils of Dioscorea batatas Decne., on photosynthetic reactions of chloroplasts from spinach (Spinacia oleracea L.) and on respiration of mitochondria from potatoes (Solanum tuberosum L.) were investigated. In chloroplasts, the batatasins effectively inhibited CO₂-dependent oxygen evolution and electron flow from water to acceptors such as dichlorophenolindophenol, ferricyanide and methylviologen. Photosystem-I dependent electron transport from ascorbate to oxygen was stimulated. The proton conductivity of thylakoid membranes was increased and phosphorylation was uncoupled from electron transport. Inhibition of electron transport with water as electron donor appeared to precede uncoupling. In mitochondrial, batatasin I did not much inhibit succinate-dependent O₂ uptake in the absence of ADP, but caused strong inhibition in the presence of ADP. Batatasins III and V inhibited oxygen uptake irrespective of the presence or absence of ADP. Inhibition of chloroplast and mitochondrial reactions by batatasins was shown to be reversible.Abbrevations B-I batatasin I, 6-hydroxy-2,4,7-trimethoxyphenanthrene - B-III batatasin III, 3,3-dihydroxy-5-methoxybibenzyl - B-V batatasin V, 2-hydroxy-3,4,5-trimethoxybibenzyl - Chl chlorophyll - MV methylviologen - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PVP polyvinylpyrrolidone     

Symplasmic and apoplasmic radial ion transport in plant roots

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of -glucan phosphorylase were visible. Using specific -glucans incorporated into the gel before electrophoresis we have tentatively correlated -amylase and -amylase with both hydrolytic activities. The activities of -glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of -glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and -glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DDT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G1P glucose 1-phosphate - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - P_i inorganic orthophosphate - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase, EC 4.1.1.39     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status.Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake.The phosphate-uptake system appeared similar in all strains with apparent K _m values ranging from 1.6 M to 6.0 M phosphate and maximum activities from 17.2 to 126 nmol · min^-1 · (mg dry weight of cells)^-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of ³²P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulphonic acid     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

5-O-β-d-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin,an inhibitor of photophosphorylation in spinach chloroplasts

5-O--d-galactopyranosyl-7-methoxy-3,4-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 M. H⁺-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg⁺²-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca⁺²-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CF_O) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.Abbreviations CF₁ chloroplast coupling factor 1 - CF_O coupling factor zero - DCCD dicyclohexylcarbodiimide - DTT dithiothreitol - EDTA ethylene-diaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-morpholino) ethanesulphonic acid - TCA trichloroacetic acid Taken in part from PhD thesis of M.R. Calera.     

Physical properties and metabolite regulation of ribulose bisphosphate carboxylase from Thiobacillus A2

Purified ribulose-bisphosphate carboxylase (EC 4.1.1.39) was strongly and equally inhibited either by ADP or GDP and to a lesser extent by IDP. AMP or ATP exerted little effect on activity. Inhibition by the nucleotide diphosphates was competitive with respect to RuBP and non-competitive with respect to CO₂ and Mg²⁺, respectively. Treatment of the enzyme with urea or guanidine-HCl resulted in rapid loss of activity that was not restored by dialysis even in the presence of Mg²⁺ and cysteine. Sodium dodecyl sulfate electrophoresis of 8.0 M urea treated enzyme revealed the presence of a fast-moving (small) sub-unit with molecular weight 14150 and a slower moving (large) sub-unit with molecular weight 68000. Examination of native enzyme by sodium dodecyl sulfate electrophoresis gave sub-units of 13700 and 55500 respectively. The amino acid content standardized to phenylalanine was essentially similar to that from other sources. Arrhenius plots showed a break at 29°C with an E _a of 12.34 kcal per mole for the steeper part of the curve and a H of 11.43 kcal per mole while for the less steep region, the E _a was 1.04 kcal per mole and the H 1.92 kcal per mole.Abbreviations ADP adenosine-5-diphosphate - AMP adenosine-5-monophosphate - ATP adenosine-5-triphosphate - CDP cytidine-5-diphosphate - CMP cytidine-5-monophosphate - CTP cytidine-5-triphosphate - FDP fructose-1,6-diphosphate - F6P fructose-6-phosphate - GDP guanosine-5-diphosphate - GMP guanosine-5-monophosphate - G6P glucose-6-phosphate - GTP guanosine-5-triphosphate - IDP inosine-5-diphosphate - IMP inosine-5-monophosphate - PEP phosphoenolpyruvate - 6PG 6-phosphogluconate - R1P ribose-1-phosphate - R5P ribose-5-phosphate - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecyl sulfate - TDP thymidine-5-diphosphate - TMP thymidine-5-monophosphate - TTP thymidine-5-triphosphate - UDP uridine-5-diphosphate - UMP uridine-5-monophosphate - UTP uridine-5-triphosphate     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).     

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3,4-diethoxyphenyl)-1,3(dihydroxy)-2-(4'-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial , bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the , bond of 1-(3-4-diethoxyphenyl)-1-(hydroxy)-2-(4'-methoxyphenyl)ethane (XI) also occurred, indicating that a hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2,2(dihydroxy)-2-(4'-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the , bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MS mass spectrometry     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.     

Very slow growth of Bacillus polymyxa: Stringent response and maintenance energy

Bacillus polymyxa grown in a recycling fermentor shows the same behavior previously observed with Escherichia coli: 3 successive growth phases. In the last 2 phases the growth rate is linear and the apparent maintenance energy demand rate and the molar growth yield are both independent of the specific growth rate, , and of the cells mass. The final phase of very slow growth is an indefinitely prolonged state of strong, stringent control, the regulatory system based on guanosine 3-diphosphate 5-diphosphate, and guanosine 3-diphosphate 5-triphosphate. The maximum cost of this stringent response is calculated to be 9% of the energy available to these energy-limited cells. There is a further energy cost contained in substantial amounts of DNA, RNA, and protein released from the cells during the latter 2 growth phases. The cost of production of these extra cellular anabolites ranges from 8–11% of the available energy.After a carbon-energy upshift in phase 3, the population growth rate immediately returned to that of early phase 2 growth, 50 h or more earlier.If maintenance energy is considered as energy expended by cells to maintain homeostasis, catabolic capacity, or anabolic potential, then the cost of stringent control — which preserves the fidelity of protein synthesis in slowly growing cells — must be considered a maintenance energy cost.Abbreviations GPR glucose provision rate - F_R medium flow rate - S_R substrate concentration - V_F fermentor volume - F_S filtrate removal rate - ppGpp guanosine 3-diphosphate 5-diphosphate - pppGpp guanosine 3-diphosphate 5-triphosphate     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

Nucleic acid metabolism in yeast

Summary The three haploid yeast strains T2tmp1-3, T2tmp1-1, and T6tmp1-51 auxotrophic for 5-dTMP differ in their requirement for thymidylate: 72, 16, and 3 g 5-dTMP/ml will restore optimal growth, respectively. Thymidylate low requirement in strain T2tmp1-1 and T6tmp1-51 is termed tlrA and tlrC, respectively. When the growth medium is made 5x10^-4 M for 5-dTMP only strain T6tmp1-51 is severely inhibited in RNA and DNA synthesis. This inhibition is reversible after removal of excessive 5-dTMP. The inhibitory characteristic is in marked contrast to thymineless death due to the lack of 5-dTMP in strain T6tmp1-51 where only DNA synthesis stops while RNA synthesis continues. The inhibitory effect of 5x10^-4 M 5-dTMP is not due to the 5-dTMP auxotrophy but to the thymidylate low requiring character (tlrC) in strain T6tmp1-51. The arrest of RNA and DNA synthesis by high concentrations of exogenous 5-dTMP suggests a regulatory role of either the monoor triphosphate on nucleoside or nucleotide biosynthesis in yeast.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase