DOCSLIB.ORG

Structural Basis of Light-Induced Redox Regulation in the Calvin–Benson Cycle in Cyanobacteria

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structural Basis of Light-Induced Redox Regulation in the Calvin–Benson Cycle in Cyanobacteria Structural basis of light-induced redox regulation in the Calvin–Benson cycle in cyanobacteria Ciaran R. McFarlanea,1, Nita R. Shaha,1, Burak V. Kabasakala,2, Blanca Echeverriaa, Charles A. R. Cottona,3, Doryen Bubecka,4, and James W. Murraya,4 aDepartment of Life Sciences, Imperial College London, SW7 2AZ, United Kingdom Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved September 12, 2019 (received for review April 18, 2019) Plants, algae, and cyanobacteria fix carbon dioxide to organic ordering the C-terminal domain (16), enabling it to bind GAPDH, carbon with the Calvin–Benson (CB) cycle. Phosphoribulokinase which is then inhibited. (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) The GAPDH-CP12 complex can then bind PRK, in an obli- are essential CB-cycle enzymes that control substrate availability gate sequential reaction (17). Previous structural studies of the for the carboxylation enzyme Rubisco. PRK consumes ATP to pro- GAPDH-CP12 complex resolved only a C-terminal fragment of duce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH CP12, so it remained unclear how CP12 regulated both GAPDH catalyzes the reduction step of the CB cycle with NADPH to pro- and PRK. A recent structure of a cyanobacterial CP12-CBS 2 duce the sugar glyceraldehyde 3-phosphate (GAP), which is used domain protein resolved an N-terminal CP12-like region; how- for regeneration of RuBP and is the main exit point of the cycle. ever, the protein does not form a ternary complex with GAPDH GAPDH and PRK are coregulated by the redox state of a condition- and PRK (8). Calvin cycle GAPDH uses NADPH physiologically ally disordered protein CP12, which forms a ternary complex with to reduce BPG, although NADH may also be a substrate. both enzymes. However, the structural basis of CB-cycle regulation CP12 is conserved in oxygenic phototrophs from cyanobacteria by CP12 is unknown. Here, we show how CP12 modulates the to plants (18). Plants often have more than 1 CP12 isoform, with activity of both GAPDH and PRK. Using thermophilic cyanobacte- tissue-specific patterns of expression (19). Plants also have rial homologs, we solve crystal structures of GAPDH with different 2 photosynthetic GAPDH isoforms GAP-A and GAP-B. GAP-A cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK com- is similar to the cyanobacterial enzyme while GAP-B has a BIOCHEMISTRY plex by electron cryo-microscopy, we reveal that formation of the C-terminal extension homologous to the C-terminal GAPDH N-terminal disulfide preorders CP12 prior to binding the PRK active binding domain of CP12 (20). GAP-A and GAP-B form an A2B2 site, which is resolved in complex with CP12. We find that CP12 tetramer, which further aggregates to A8B8 on oxidation of the binding to GAPDH influences substrate accessibility of all GAPDH GAP-B CP12-like region (20). Information from cyanobacteria is active sites in the binary and ternary inhibited complexes. Our still applicable to plants, as the cyanobacterial CP12 system is structural and biochemical data explain how CP12 integrates re- a subset of the plant regulatory system. The lack of structural sponses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. Significance redox regulation | carbon fixation | photosynthesis | Calvin–Benson cycle The Calvin–Benson (CB) cycle in plants, algae, and cyanobac- teria fixes most of the carbon in most of the biomass on Earth. lants regulate their carbon fixation and other reactions by The CB cycle is regulated by the redox state, which enables it to Predox state using it as a proxy for light (1). The product of the be turned off in the dark. One part of this regulatory system is photosynthetic electron transport chain is reduced ferredoxin, the small protein CP12, which binds to 2 essential CB-cycle which is used to produce NADPH in the cyanobacterial cyto- enzymes in the dark, inactivating them. We have solved the plasm or chloroplast stroma. In the light, the chloroplast stroma structure of the complex between CP12 and the enzymes, is reducing, and in the dark, or when the light reactions are explaining the mechanism of deactivation. Now that this is otherwise inactive, the stroma becomes oxidizing. Thioredoxin understood, this structure can be used as the starting point for (2) proteins are reduced by ferredoxin and can then exchange a modulating the redox regulation, which may have applications disulfide with a target. Example targets are the sedoheptulose (3) in improving crop productivity. and fructose bisphosphatases (4), which have pairs of cysteines that oxidize to form disulfide bonds that inactivate the enzyme. Author contributions: C.R.M., D.B., and J.W.M. designed research; C.R.M., N.R.S., B.V.K., The carbon-fixation reaction is catalyzed by Rubisco, which B.E., C.A.R.C., D.B., and J.W.M. performed research; C.R.M., N.R.S., D.B., and J.W.M. ana- carboxylates ribulose bisphosphate (RuBP). RuBP is produced lyzed data; and C.R.M., N.R.S., D.B., and J.W.M. wrote the paper. by phosphoribulokinase (PRK) in the final regeneration reaction, The authors declare no competing interest. in which a phosphate group is transferred from ATP to ribulose 5- This article is a PNAS Direct Submission. phosphate (5). Plant-type PRK is dimeric and has 2 conserved This open access article is distributed under Creative Commons Attribution-NonCommercial- disulfide bonds, one at the N terminus and the other near the C NoDerivatives License 4.0 (CC BY-NC-ND). terminus close to the dimer interface (6, 7). The N-terminal disulfide Data deposition: Crystallography, atomic coordinates, and structure factors have been – deposited in the PDB and EMDB repositories: PDB IDs 6GFP, 6GFQ, 6GFR, 6GFO, 6GG7, bond inactivates most of the activity when formed (8 10), but may 6GHR, 6GHL, and 6GVE; EMDB ID EMD0071. be reduced in plants by thioredoxin f (11). 1C.R.M. and N.R.S. contributed equally to this work. The reduction step of the Calvin–Benson (CB) cycle is cata- 2Present address: School of Biochemistry, University of Bristol, BS8 1TD Bristol, lyzed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), United Kingdom. which uses NADPH to reduce bisphosphoglycerate (BPG) to 3Present address: Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam- glyceraldehyde 3-phosphate (GAP), the main product exit point Golm, Germany. of the cycle (12). In cyanobacteria, GAPDH has no disulfide 4To whom correspondence may be addressed. Email: [email protected] or j.w. bonds, instead redox regulation of GAPDH is driven by 2 disulfide [email protected]. bonds in the small (∼8 kDa) regulatory inhibitor protein CP12 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (13, 14), which is disordered under reducing conditions (15). 1073/pnas.1906722116/-/DCSupplemental. Under oxidizing conditions, 2 disulfide bonds form in CP12, www.pnas.org/cgi/doi/10.1073/pnas.1906722116 PNAS Latest Articles | 1of7 Downloaded by guest on September 28, 2021 information for full-length CP12 and CP12-bound regulatory CP12 binding to GAPDH mutually exclusive. CP12-Glu69 is not complexes has prevented understanding of a key mechanism of only important for dinucleotide selection, but is also essential for CB-cycle redox regulation. To understand, at a molecular level, ternary complex formation in Chlamydomonas (24). how the CB cycle is redox regulated in response to light, we solved crystal structures of a thermophilic cyanobacterial GAPDH with Structure of Full-Length CP12 Bound to GAPDH. In 3 conditions, full-length CP12 and built an atomic model of the entire cyano- GAPDH-CP12 crystallized with one or more full-length CP12 bacterial GAPDH-CP12-PRK ternary complex using electron visible in the electron density, at a best resolution of 2.1 Å. We cryo-microscopy (cryoEM). obtained 4 crystallographically independent full-length oxidized CP12 molecules bound to GAPDH. CP12 comprises an N-terminal Results and Discussion PRK binding domain (residues 1–52) and a C-terminal GAPDH – Structures of GAPDH with Different Nucleotides Bound and CP12 binding domain (residues 55 75) connected by a fully flexible C-Terminal Regions. A previous structure of cyanobacterial linker (Fig. 2). If the C terminus of CP12 is bound to GAPDH, GAPDH bound to CP12 had 4 CP12 per GAPDH tetramer (21), the N-terminal domain can adopt any relative angle. Although but the stoichiometry for eukaryotic complexes suggested by the relative orientations of the N- and C-terminal domains of structure (22) and biochemistry (23) was 2. Working with recombinant CP12 vary in our structures, the structures of both domains are conserved across 4 crystallographically independent CP12 mol- proteins from the thermophilic cyanobacterium Thermosynechococcus ecules (Fig. 2 C and D). elongatus, we found the cyanobacterial GAPDH -CP12 complex was 4 2 The CP12 N-terminal PRK binding domain is a 2-helix bundle, stable to gel-filtration and crystallized with this stoichiometry. We stabilized by a disulfide bridge between Cys-19 and 29 near the call the 2 CP12-occupied active sites proximal and the 2 un- helical turn. The 2 helices bury a small hydrophobic core, including occupied sites distal. In all our structures, and those published, the a short leucine zipper. Helix-2 (27-52) contains the conserved 2 proximal sites are in equivalent positions in the GAPDH tet- CP12 characteristic motif, AWDA(V/L)EEL (Figs. 2B and 3E) ramer. We obtained a GAPDH4-CP124 structure when GAPDH (25), which forms an acidic patch on the surface. was incubated with 10-fold excess CP12 (SI Appendix,Fig.S1), so the physiological GAPDH-CP12 complex was unclear (21, 22). Structure of GAPDH-CP12-PRK Complex. We solved the structure of Only the C terminus of CP12 was visible in these structures.
Load more

Recommended publications
  • Chapter 4. Enzyme Kinetics
    Chapter 4. Enzyme Kinetics Enzyme Engineering 4.1 Why is enzyme kinetics important? 1. It provides valuable information for enzyme mechanism 2. It gives an insight into the role of an enzyme under physiological conditions 3. It can help show how the enzyme activity is controlled and regulated 1 4.2 How to obtain enzyme kinetics? Measure the formation of product or disappearance of substrate Mg2+ D-Glucose + ATP D-Glucose-6-phosphate + ADP 1. Discontinuous method Add the enzyme, then stop the reaction at different time by adding acid to the sample Measure the concentration of substrate or product using HPLC or other chromatography 4.2 How to obtain enzyme kinetics? 2. Continuous method Mg2+ D-Glucose + ATP D-Glucose-6-phosphate + ADP NADP+ NADPH D-Glucono-δ-lactone 6-phosphate + ADP NADPH absorb at 340nm, while NADP+ does not If a coupled reaction is used, the second reaction should not be rate-limiting (sufficient amount of the enzyme must be provided) Continuous method is not always available 2 4.2 How to obtain enzyme kinetics? Radioactive or fluorescent-labeling is useful Precautions The substrate, buffers, etc. must be extremely pure Enzyme should be pure Enzyme should be stable as the assay is proceeded * Temp, pH must be maintained carefully Once steady state is achieved, reaction rate must be constant and proportional to the enzyme added Initial rate of reaction should be measured to avoid possible complexity 4.3 How to analyze kinetic data? 4.3.1 One substrate reactions k 1 k2 E + S ES E + P k-1 Equilibrium assumption (1913) Second reaction is slower than first reverse reaction (k-1 >> k2) Vmax[S] V0 = Michaelis-Menten eqn K s +[S] [E][S] Vmax = k2[E]0 K = s [ES] 3 4.3 How to analyze kinetic data? Steady state assumption (1925) [ES] is constant Vmax[S] V0 = Km +[S] Vmax = k2[E]0 k2 + k−1 Km = k1 4.3.1 One substrate reactions Lineweaver-Burk equation 1 K 1 1 = m + v [S] Vmax Vmax Eddie-Hofstee equation v V v = max − [S] Km Km 4 4.3.1 One substrate reactions Significance of the result 1.
    [Show full text]
  • The Thioredoxin Trx-1 Regulates the Major Oxidative Stress Response Transcription Factor, Skn-1, in Caenorhabditis Elegans
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2016 THE THIOREDOXIN TRX-1 REGULATES THE MAJOR OXIDATIVE STRESS RESPONSE TRANSCRIPTION FACTOR, SKN-1, IN CAENORHABDITIS ELEGANS Katie C. McCallum Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Cellular and Molecular Physiology Commons, Medicine and Health Sciences Commons, Molecular Genetics Commons, and the Organismal Biological Physiology Commons Recommended Citation McCallum, Katie C., "THE THIOREDOXIN TRX-1 REGULATES THE MAJOR OXIDATIVE STRESS RESPONSE TRANSCRIPTION FACTOR, SKN-1, IN CAENORHABDITIS ELEGANS" (2016). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 655. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/655 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. THE THIOREDOXIN TRX-1 REGULATES THE MAJOR OXIDATIVE STRESS RESPONSE TRANSCRIPTION FACTOR, SKN-1, IN CAENORHABDITIS ELEGANS A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Katie Carol McCallum, B.S.
    [Show full text]
  • Indications for a Central Role of Hexokinase Activity in Natural Variation of Heat Acclimation in Arabidopsis Thaliana
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 June 2020 doi:10.20944/preprints202006.0169.v1 Article Indications for a central role of hexokinase activity in natural variation of heat acclimation in Arabidopsis thaliana Vasil Atanasov §, Lisa Fürtauer § and Thomas Nägele * LMU Munich, Plant Evolutionary Cell Biology, Großhaderner Str. 2-4, 82152 Planegg, Germany § Authors contributed equally * Correspondence: [email protected] Abstract: Diurnal and seasonal changes of abiotic environmental factors shape plant performance and distribution. Changes of growth temperature and light intensity may vary significantly on a diurnal, but also on a weekly or seasonal scale. Hence, acclimation to a changing temperature and light regime is essential for plant survival and propagation. In the present study, we analyzed photosynthetic CO2 assimilation and metabolic regulation of the central carbohydrate metabolism in two natural accessions of Arabidopsis thaliana originating from Russia and south Italy during exposure to heat and a combination of heat and high light. Our findings indicate that it is hardly possible to predict photosynthetic capacities to fix CO2 under combined stress from single stress experiments. Further, capacities of hexose phosphorylation were found to be significantly lower in the Italian than in the Russian accession which could explain an inverted sucrose-to-hexose ratio. Together with the finding of significantly stronger accumulation of anthocyanins under heat/high light these observations indicate a central role of hexokinase activity in stabilization of photosynthetic capacities within a changing environment. Keywords: photosynthesis; carbohydrate metabolism; hexokinase; heat acclimation; environmental changes; natural variation; high light; combined stress. 1. Introduction Changes of growth temperature and light intensity broadly affect plant molecular, physiological and developmental processes.
    [Show full text]
  • METABOLIC EVOLUTION in GALDIERIA SULPHURARIA By
    METABOLIC EVOLUTION IN GALDIERIA SULPHURARIA By CHAD M. TERNES Bachelor of Science in Botany Oklahoma State University Stillwater, Oklahoma 2009 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY May, 2015 METABOLIC EVOLUTION IN GALDIERIA SUPHURARIA Dissertation Approved: Dr. Gerald Schoenknecht Dissertation Adviser Dr. David Meinke Dr. Andrew Doust Dr. Patricia Canaan ii Name: CHAD M. TERNES Date of Degree: MAY, 2015 Title of Study: METABOLIC EVOLUTION IN GALDIERIA SULPHURARIA Major Field: PLANT SCIENCE Abstract: The thermoacidophilic, unicellular, red alga Galdieria sulphuraria possesses characteristics, including salt and heavy metal tolerance, unsurpassed by any other alga. Like most plastid bearing eukaryotes, G. sulphuraria can grow photoautotrophically. Additionally, it can also grow solely as a heterotroph, which results in the cessation of photosynthetic pigment biosynthesis. The ability to grow heterotrophically is likely correlated with G. sulphuraria ’s broad capacity for carbon metabolism, which rivals that of fungi. Annotation of the metabolic pathways encoded by the genome of G. sulphuraria revealed several pathways that are uncharacteristic for plants and algae, even red algae. Phylogenetic analyses of the enzymes underlying the metabolic pathways suggest multiple instances of horizontal gene transfer, in addition to endosymbiotic gene transfer and conservation through ancestry. Although some metabolic pathways as a whole appear to be retained through ancestry, genes encoding individual enzymes within a pathway were substituted by genes that were acquired horizontally from other domains of life. Thus, metabolic pathways in G. sulphuraria appear to be composed of a ‘metabolic patchwork’, underscored by a mosaic of genes resulting from multiple evolutionary processes.
    [Show full text]
  • Labeled in Thecourse of Glycolysis, Since Phosphoglycerate Kinase
    THE STATE OF MAGNESIUM IN CELLS AS ESTIMATED FROM THE ADENYLATE KINASE EQUILIBRIUM* BY TRWIN A. RoSE THE INSTITUTE FOR CANCER RESEARCH, PHILADELPHIA Communicated by Thomas F. Anderson, August 30, 1968 Magnesium functions in many enzymatic reactions as a cofactor and in com- plex with nucleotides acting as substrates. Numerous examples of a possible regulatory role of Mg can be cited from studies with isolated enzymes,'- and it is known that Mg affects the structural integrity of macromolecules such as trans- fer RNA" and functional elements such as ribosomes.'0 The major problem in translating this information on isolated preparations to the functioning cell is the difficulty in determining the distribution of Mg and the nucleotides among the free and complexed forms that function in the region of the cell for which this information is desired. Nanningall based an attempt to calculate the free Mg2+ and Ca2+ ion concentrations of frog muscle on the total content of these metals and of the principal known ligands (adenosine 5'-triphosphate (ATP), creatine-P, and myosin) and the dissociation constants of the complexes. However, this method suffers from the necessity of evaluating the contribution of all ligands as well as from the assumption that all the known ligands are contributing their full complexing capacity. During studies concerned with the control of glycolysis in red cells and the control of the phosphoglycerate kinase step in particular, it became important to determine the fractions of the cell's ATP and adenosine 5'-diphosphate (ADP) that were present as Mg complexes. Just as the problem of determining the distribution of protonated and dissociated forms of an acid can be solved from a knowledge of pH and pKa of the acid, so it would be possible to determine the liganded and free forms of all rapidly established Mg complexes from a knowledge of Mg2+ ion concentration and the appropriate dissociation constants.
    [Show full text]
  • Catalase Kinetics
    Experiment #4: Catalase Kinetics MASSACHUSETTS INSTITUTE OF TECHNOLOGY Department of Chemistry 5.310 Laboratory Chemistry 1 EXPERIMENT #4 A Study of the Kinetics of the Enzyme Catalase and its Reaction 2 3 With H2O2. A Further Study on Protein Assay Quantitation of Catalase I. OVERVIEW OF THE EXPERIMENT In this experiment, the student will investigate the enzyme activity of catalase by studying the decomposition of H2O2 to form water and oxygen. Using an oxygen based pressure sensor the student measures the amount of oxygen produced and then calculates the rate of the enzyme catalyzed reaction under various conditions. The student also completes a Protein Assay on an unknown sample of catalase using the Coomassie® Plus Protein Assay from Pierce to determine the protein concentration of the sample. The correlation between the actual and calculated concentration gives an indication of the experimental skills used in carrying out the experiment. II. OBJECTIVES This is an integrated experiment that includes topics from physical chemistry and biochemistry. It is designed to introduce students to the basics of: • Studying the effects of reaction environment, including temperature, and varying substrate concentration on the rate of an enzyme-catalyzed reaction. • Combining physical chemistry and biochemistry, principles and theory, with the goal of determining various biochemical and biophysical constants for the enzyme catalase. • How to acquire experimental kinetic data for an enzyme catalyzed reaction. • Learning how to perform data manipulation in order to extract out information such as rate constants from experimental kinetic data. • Correct handling of UV-VIS Spectroscopy 1 This experiment was synthesized by John J.
    [Show full text]
  • Estimation of the Overall Kinetic Parameters of Enzyme Inactivation Using an Isoconversional Method D
    Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan To cite this version: D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan. Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method. Biophysical Chemistry, Elsevier, 2008, 138 (1-2), pp.50. 10.1016/j.bpc.2008.09.003. hal-00501718 HAL Id: hal-00501718 https://hal.archives-ouvertes.fr/hal-00501718 Submitted on 12 Jul 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ÔØ ÅÒÙ×Ö ÔØ Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan PII: S0301-4622(08)00176-2 DOI: doi: 10.1016/j.bpc.2008.09.003 Reference: BIOCHE 5155 To appear in: Biophysical Chemistry Received date: 17 August 2008 Revised date: 2 September 2008 Accepted date: 3 September 2008 Please cite this article as: D. Oancea, Alexandrina Stuparu, Madalina Nita, Mi- haela Puiu, Adina Raducan, Estimation of the overall kinetic parameters of en- zyme inactivation using an isoconversional method, Biophysical Chemistry (2008), doi: 10.1016/j.bpc.2008.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication.
    [Show full text]
  • Determination of Enzyme Kinetics in the Eppendorf Biospectrometer® Kinetic Using a Hexokinase and Glucose-6- Phosphate-Dehydrogenase from Saccharomyces Cerevisiae
    USERGUIDE No. 39 I Detection I September 2014 Determination of enzyme kinetics in the Eppendorf BioSpectrometer® kinetic using a hexokinase and glucose-6- phosphate-dehydrogenase from Saccharomyces cerevisiae Martin Armbrecht, Eppendorf AG, Hamburg, Germany Abstract This Userguide will demonstrate measurement of enzyme activity using the Eppendorf BioSpectrometer. In order to optimize the measurement process, preliminary measurements were performed using the method “single continuous”. The actual activity measurements were then performed based on these results. Enzyme activities were determined via linear regression. For the measurements, a coupled reaction of a hexokinase and a glucose-6-phosphate-dehydrogenase from the baker’s yeast ‚ was used. Since the Eppendorf BioSpectrometer kinetic is equipped with a temperature controlled cuvette chamber, temperature dependence of this reaction could also be demonstrated. The highest activity was detected at 37 °C. Introduction Metabolic significance of the hexokinase reaction Activation of glucose via the hexokinase reaction Glycolysis Glucose degradation thus sustains respiration of all living beings. One central area within the metabolism of nearly all living beings is Prior to degradation, glucose needs to be rendered susceptible for the enzymatic degradation of glucose in the cytosol, or cytoplasma, degradation, i.e., activated. This process involves phosphorylation by respectively, in their cells. This process is called glycolysis. Glucose a hexokinase; with the expenditure of ATP, glucose-6-phosphate is is converted to 2 molecules of pyruvate in a stepwise fashion. One produced. Thus, the latter is the actual initial substrate of glycolysis molecule of glucose yields 2 ATP and 2 redox equivalents in the form (fig.1). In addition, glucose-6-phosphate is the initial substrate of a further of NADPH2.
    [Show full text]
  • New Nucleotide Sequence Data on the EMBL File Server
    .=) 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 2 413 New nucleotide sequence data on the EMBL File Server October 30, 1990 to November 13, 1990 New nucleotide sequence data, available from the EMBL File Server, (see Stoehr, P.J. and Omond, R.A. (1989) Nucleic Acids Res., 17 (16), 6763 -6764), are reported below. Availability of all the newest sequence data is the result of collaboration between.the EMBL Data Library and GenBank' , and data are supplied regularly by both groups. Updates to existing data are not reported here. This report has been prepared by the EMBL Data Library. PRIMATES: Human casein kinase II alpha subunit mRNA, complete cds. Human specific HS5 DNA Lozeman F.J., Litchfield D.W., Piening C., Takio K., Walsh Ueda S., Washio K., Kurosaki K.; K.A., Krebs E.G.; Genomics 8:7-12(1990). X17579 Biochemistry 29:8436-8447(1990). M55268 Human mRNA for heat shock protein HSP27 Human mRNA for actin-binding protein (ABP-280) Carper S.W., Rocheleau T.A., Storm F.K.; Gorlin J.B., Yamin R., Egan S., Stewart M., Stossel T.P., Nucleic Acids Res. 18:6457-6457(1990). X54079 Kwiatkowski D.J., Hartwig J.H.; J. Cell Biol. 111:1089-1105(1990). X53416 Human Ig germline H-chain D-region Dxpl and Dxp'1 genes, 3' Human amiloride-binding protein, complete cds. end. Barbry P., Champe M., Chassande O., Munemitsu S., Champigny Huang C., Stollar B.David.; G., Lingueglia E., Maes P., Frelin C., Tartar A., Ullrich A., Unpublished. M37485 Lazdunski M.; Proc. Natl. Acad.
    [Show full text]
  • Supplementary Materials
    Supplementary Materials Figure S1. Differentially abundant spots between the mid-log phase cells grown on xylan or xylose. Red and blue circles denote spots with increased and decreased abundance respectively in the xylan growth condition. The identities of the circled spots are summarized in Table 3. Figure S2. Differentially abundant spots between the stationary phase cells grown on xylan or xylose. Red and blue circles denote spots with increased and decreased abundance respectively in the xylan growth condition. The identities of the circled spots are summarized in Table 4. S2 Table S1. Summary of the non-polysaccharide degrading proteins identified in the B. proteoclasticus cytosol by 2DE/MALDI-TOF. Protein Locus Location Score pI kDa Pep. Cov. Amino Acid Biosynthesis Acetylornithine aminotransferase, ArgD Bpr_I1809 C 1.7 × 10−4 5.1 43.9 11 34% Aspartate/tyrosine/aromatic aminotransferase Bpr_I2631 C 3.0 × 10−14 4.7 43.8 15 46% Aspartate-semialdehyde dehydrogenase, Asd Bpr_I1664 C 7.6 × 10−18 5.5 40.1 17 50% Branched-chain amino acid aminotransferase, IlvE Bpr_I1650 C 2.4 × 10−12 5.2 39.2 13 32% Cysteine synthase, CysK Bpr_I1089 C 1.9 × 10−13 5.0 32.3 18 72% Diaminopimelate dehydrogenase Bpr_I0298 C 9.6 × 10−16 5.6 35.8 16 49% Dihydrodipicolinate reductase, DapB Bpr_I2453 C 2.7 × 10−6 4.9 27.0 9 46% Glu/Leu/Phe/Val dehydrogenase Bpr_I2129 C 1.2 × 10−30 5.4 48.6 31 64% Imidazole glycerol phosphate synthase Bpr_I1240 C 8.0 × 10−3 4.7 22.5 8 44% glutamine amidotransferase subunit Ketol-acid reductoisomerase, IlvC Bpr_I1657 C 3.8 × 10−16
    [Show full text]
  • Domain Structure of the Glucocorticoid Receptor Protein
    Proc. Nati. Acad. Sci. USA Vol. 84, pp. 4437-4440, July 1987 Biochemistry Domain structure of the glucocorticoid receptor protein (proteolysis/steroid binding/DNA binding/protein sequence) JAN CARLSTEDT-DUKE*, PER-ERIK STROMSTEDT*, ORJAN WRANGEt, TOMAS BERGMANt, JAN-AKE GuSTAFSSON*, AND HANS JORNVALLf *Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, F69, S-141 86 Huddinge, Sweden; and Departments of tMedical Cell Genetics and tChemistry, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden Communicated by Viktor Mutt, March 26, 1987 (receivedfor review December 1, 1986) ABSTRACT The purified rat liver glucocorticoid receptor GR was eluted with 27.5 mM MgCl2 and further purified by protein was analyzed by limited proteolysis and amino acid chromatography on DEAE-Sepharose, eluted with a linear sequence determination. The NH2 terminus appears to be NaCl gradient. The receptor was detected by analysis for 3H blocked. The steroid-binding domain, defined by a unique radioactivity. Each purification resulted in a yield of '50 ,g tryptic cleavage site, corresponds to the COOH-terminal part of GR, starting from eight rat livers. The purified prepara- of the protein with the domain border in the region of residue tions of GR contained the 94-kDa GR, the 72-kDa GR- 518. The DNA-binding domain, defined by a region with associated protein that is unrelated to GR (12, 13), as well as chymotryptic cleavage sites, is immediately adjacent to the very small amounts of proteolytic GR fragments (usually steroid-binding domain and reflects another domain border in <5% of total protein according to densitometric analysis of the region of residues 410-414.
    [Show full text]
  • Table S1. List of Oligonucleotide Primers Used
    Table S1. List of oligonucleotide primers used. Cla4 LF-5' GTAGGATCCGCTCTGTCAAGCCTCCGACC M629Arev CCTCCCTCCATGTACTCcgcGATGACCCAgAGCTCGTTG M629Afwd CAACGAGCTcTGGGTCATCgcgGAGTACATGGAGGGAGG LF-3' GTAGGCCATCTAGGCCGCAATCTCGTCAAGTAAAGTCG RF-5' GTAGGCCTGAGTGGCCCGAGATTGCAACGTGTAACC RF-3' GTAGGATCCCGTACGCTGCGATCGCTTGC Ukc1 LF-5' GCAATATTATGTCTACTTTGAGCG M398Arev CCGCCGGGCAAgAAtTCcgcGAGAAGGTACAGATACGc M398Afwd gCGTATCTGTACCTTCTCgcgGAaTTcTTGCCCGGCGG LF-3' GAGGCCATCTAGGCCATTTACGATGGCAGACAAAGG RF-5' GTGGCCTGAGTGGCCATTGGTTTGGGCGAATGGC RF-3' GCAATATTCGTACGTCAACAGCGCG Nrc2 LF-5' GCAATATTTCGAAAAGGGTCGTTCC M454Grev GCCACCCATGCAGTAcTCgccGCAGAGGTAGAGGTAATC M454Gfwd GATTACCTCTACCTCTGCggcGAgTACTGCATGGGTGGC LF-3' GAGGCCATCTAGGCCGACGAGTGAAGCTTTCGAGCG RF-5' GAGGCCTGAGTGGCCTAAGCATCTTGGCTTCTGC RF-3' GCAATATTCGGTCAACGCTTTTCAGATACC Ipl1 LF-5' GTCAATATTCTACTTTGTGAAGACGCTGC M629Arev GCTCCCCACGACCAGCgAATTCGATagcGAGGAAGACTCGGCCCTCATC M629Afwd GATGAGGGCCGAGTCTTCCTCgctATCGAATTcGCTGGTCGTGGGGAGC LF-3' TGAGGCCATCTAGGCCGGTGCCTTAGATTCCGTATAGC RF-5' CATGGCCTGAGTGGCCGATTCTTCTTCTGTCATCGAC RF-3' GACAATATTGCTGACCTTGTCTACTTGG Ire1 LF-5' GCAATATTAAAGCACAACTCAACGC D1014Arev CCGTAGCCAAGCACCTCGgCCGAtATcGTGAGCGAAG D1014Afwd CTTCGCTCACgATaTCGGcCGAGGTGCTTGGCTACGG LF-3' GAGGCCATCTAGGCCAACTGGGCAAAGGAGATGGA RF-5' GAGGCCTGAGTGGCCGTGCGCCTGTGTATCTCTTTG RF-3' GCAATATTGGCCATCTGAGGGCTGAC Kin28 LF-5' GACAATATTCATCTTTCACCCTTCCAAAG L94Arev TGATGAGTGCTTCTAGATTGGTGTCggcGAAcTCgAGCACCAGGTTG L94Afwd CAACCTGGTGCTcGAgTTCgccGACACCAATCTAGAAGCACTCATCA LF-3' TGAGGCCATCTAGGCCCACAGAGATCCGCTTTAATGC RF-5' CATGGCCTGAGTGGCCAGGGCTAGTACGACCTCG
    [Show full text]