DOCSLIB.ORG

Covalent Attachment of Diethylstilbestrol to Glutamate Dehydrogenase: Implications for Allosteric Regulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Covalent Attachment of Diethylstilbestrol to Glutamate Dehydrogenase: Implications for Allosteric Regulation Proc. Nat. Acad. Sci. USA Vol. 68, No. 5, pp. 916-919, May 1971 Covalent Attachment of Diethylstilbestrol to Glutamate Dehydrogenase: Implications for Allosteric Regulation JOHN KALLOS* AND K. P. SHAW The Research Laboratory, Department of Pathology, St. Mary's Hospital, Montreal, Canada Communicated by C. B. Anfinsen, December 17, 1970 ABSTRACT An affinity labeling reagent for the estro- glutamate dehydrogenase by means of site-specific reagents genic-binding site of bovine liver L-glutamate dehydro- and to be published). The present paper deals genase (EC 1.4.1.3) was prepared by conversion of diethyl- (9, manuscript stilbestrol to its alkylating analogue, bromoacetyldiethyl- with the covalent attachment of diethylstilbestrol to GDH stilbestrol. Under standard assay conditions, the analogue and its implication in allosteric transition. With regard to the acted as a reversible allosteric ligand with regulatory problem of allosteric transition, the present paper describes activity much like that of diethylstilbestrol. However, experiments pertinent to the following question: (a) Since incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in allosteric ligands exert a controlling function, without them- glutamate (X form) and an increase in alanine (Y form) selves participating in the reaction, will covalent attachment activities, and in covalent attachment of diethylstilbestrol of ligand to the protein produce the same, similar, or different in the ratio of 1 mol per subunit (of particle weight effects as the reversible ligand-protein interaction? (b) What is 52,000). The brominated analogue behaved as an affinity the degree of conformational stabilization resulting from label that mimicked the allosteric effects of diethyl- stilbestrol. Diethylstilbestrol protection of the enzyme permanently freezing the protein in one form as a result of the against alkylation by bromoacetylated sterol suggested chemical attachment of the ligand to the protein? competition for the same binding site, while ADP protec- Preliminary studies with various DES derivatives indicated tion indicated a shift of protein equilibrium into the X that one of the two hydroxyl groups can be modified by acet- form. The diethylstilbestrol-enzyme compound was de- sensitized (relative to the native enzyme) to allosteric ylation without loss of the capacity to bind to GDH. This ob- reagents such as ADP and GTP. The results were con- servation prompted us to attach a bromoacetyl group to one of sistent with conformational freezing of the modified the hydroxyl groups of the DES molecule. The close structural protein molecule into the Y form. analogy between bromoacetyl and acetyl DES led us to the expectation that a reversible ligand-protein interaction should the structure the Estrogenic hormones alter and catalytic produce a reversible allosteric effect, and that such a complex activity of crystalline glutamate dehydrogenase (EC 1.4.1.3) could be induced to form a covalent bond (by elimination of (GDH); they simultaneously inhibit glutamate and stimulate bromoacetic acid) and attach the DES molecule to the protein. alanine dehydrogenase activity of the enzyme (1-3). ADP can reverse the effect of these steroids (4). The action of regulatory molecules, such as diethylstilbestrol (DES), GTP, and ADP, is MATERIALS AND METHODS explained by a shift of the equilibrium between two different forms (X=Y) of the enzyme. The X form possesses high Bovine liver -glutamate dehydrogenase (type 1, ammonium glutamate activity; the Y form possesses alanine dehydro- sulfate suspension), DPNH, ADP, and GTP were obtained genase activity. Estrogen and GTP favor the Y form, while from Sigma Chemical Co. The enzyme suspension was usually ADP shifts the equilibrium to the X form. Such behavior has centrifuged before use, and the crystals were redissolved in an been rationalized in terms of allosteric interaction by Monod appropriate buffer. The enzyme solution was equilibrated with et al. (5) and Koshland (6); the term allosteric is now generally buffer either by gel filtration through Sephadex G-25 or by employed in connection with regulatory effects that are due to dialysis. Protein concentration was measured at 278 nm on the conformational changes in the protein molecule induced by a assumption that 1 mg/ml has an absorbance of 1 in a 1.0-cm ligand (7, 8). The precise molecular mechanism by which the cuvette. DES was obtained from Mann Research Laboratories allosteric ligand acts remains obscure. Clearly, more specific (New York, N.Y.). Bromoacetyldiethylstilbestrol (BADES) information is needed about the chemistry of the regulatory was prepared in our laboratory and dissolved in acetonitrile- and active sites before allosteric regulation is understood. water-ethylene glycol 2: 1: 1 before use. During the past several years we have been engaged in a Tritium-labeled BADES was prepared in our laboratory systematic chemical study of the active and regulatory sites of from ['HIDES (400 Ci/mol, Amersham/Searle Corp.). All radioactive materials were mixed with Bray's solution or toluene-2,5-diphenyloxazole solution and counted in a Nu- Abbreviations: DES, diethylstilbestrol; BADES, bromoacetyl- clear-Chicago liquid scintillation counter. All the buffer salts diethylstilbestrol; GDH, glutamate dehydrogenase. used were of analytical grade. in a * Present address: Department of Cell Biology, Faculty of Medi- Enzyme assays were performed at room temperature cine, Centre Hospitalier Universitaire, Sherbrooke, P.Q., Canada. Bausch and Lomb 505 spectrophotometer; the disappearance To whom request for reprints should be addressed. of DPNH at 340 nm was measured in 1-cm quartz cuvettes. 916 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Modification of Glutamate Dehydrogenase 917 c 0 0.3 c f._ 0 a c a E 0.5 Alanine Reaction E 4 an < E 0 E 04 0.2 >, t. a.o 0-E -U 0 -U 0.3 0 0 -a <t 0) 0.1 a -ft0 4. on Glutamate Reaction a ay 0.1I CL Concn of steroid FIG. 1. Effects of concentration of DES and BADES on GDH activity. GDH activity was measured in the assay mixture described in Methods; the GDH concentration was 0.2 jug/ml. The mixture for determining alanine dehydrogenase activity contained 0.1 M Tris (pH 8.6) and 5 X 10-2 M pyruvate in lieu of the a-ketoglutarate; the enzyme concentration was 5.15 pg/ml. Activity is expressed as A340 units per cuvette per 2 min. The reactions were started by the addition of the enzyme; Finally, it was dissolved in 0.25 ml of Nuclear-Chicago 2-min incubations were used for calculations of enzymatic solubilizer and counted in a liquid scintillation spectrometer. activity. GDH activity was measured, in the direction of a-keto- Hydroxylamine treatment of ['HJDES-GDH glutarate reductive amination, with a solution containing 1 ml Three samples, 1 ml each, were incubated at 220C; they con- of 0.05 M phosphate (pH 7.8); 1 X 10-4 M EDTA; 2 X tained [8H]DES-GDH (0.38 mg/ml), 0.15 M hydroxyl- 10-4 M DPNH; 0.2 M NH4Cl; and 2.5 X 10-2 M a-ketc- amine-HCl, 0.025 M Tris, and 0.1 M KCl (pH 7.8). The reac- glutarate. Alanine dehydrogenase activity was measured, tion was stopped by the addition of ice-cold 2 M HCl to pH 3 in the direction of pyruvate reductive amination, with a and the free [3HIDES was extracted with ether. The ether solution containing 1 ml of 0.1 M Tris * HCl (pH 8.6); 1 X 10-4 layer was washed with diluted NaHCO3, dried over MgSO4, M EDTA; 2 X 10-4 M DPNH; 0.2 M NH3Cl; and 5 X 10-2 M pyruvate. I - 100 100 > 0-e 0 Chemical reaction of GDH with BADES 5' 0 GDH (0.3 mg) was incubated in 1 ml of a reaction mixture _ 80 80 c containing 0.05 M phosphate buffer (pH 7.6), 5 X 10-5 M BADES, and 1 X 10-3 M DPNH at room temperature. The iO rate of irreversible inhibition was determined by assay of '04 60 60 o 0~ 0 aliquots. The reaction was terminated by gelfiltration through to )) a G-25 column X 40 the product was 0 Sephadex (1.5 cm); 0' C eluted with 0.05 M phosphate buffer (pH 7.6). 0C Preparation of [SH]DES-GDH .Ad 20 0 20 GDH (6 mg) was incubated in a 10-ml reaction mixture con- E taining 0.05 M phosphate (pH 7.6), 8 X 10-5M bromoacetyl- 0 O-I ['H]DES (536,000 cpm/mg), and 1 X 10-3 M DPNH: after url 30 60 90120-c 2 hr of incubation, an aliquot (5 ml) of the reaction mixture Time in Minutes was passed through a Sephadex G-25 (fine) column (2.5 X 40 to remove the excess For an FrG. 2. Progress of the chemical reaction of GDH with cm) reagent. counting, aliquot (1 BADES. GDH (0.3 mg/ml) was incubated in phosphate buffer ml) of modified protein was mixed with 10 ml of Bray's solution (0.05 M, pH 7.6) with 5 X 10-4 M BADES in the presence of 1 X and counted. Another aliquot (2 ml) of modified protein was 10-3 M DPNH. Aliquots were taken at various time intervals precipitated with 4 ml of trichloroacetic acid (30%), then and passed through a column of Sephadex G-25, and the alanine centrifuged at 5000 rpm for 15 min. The precipitate was and glutamate activities were determined. 0, Alanine; A, gluta- washed four times with absolute alcohol and once with ether. mate. Downloaded by guest on September 25, 2021 918 Biochemistry: Kallos and Shaw Proc.
Load more

Recommended publications
  • Insights Into the Molecular Basis for Substrate Binding and Specificity of the Wild-Type L-Arginine/Agmatine Antiporter Adic
    Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC Hüseyin Ilgüa,b,1, Jean-Marc Jeckelmanna,b,1, Vytautas Gapsysc, Zöhre Ucuruma,b, Bert L. de Grootc, and Dimitrios Fotiadisa,b,2 aInstitute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; bSwiss National Centre of Competence in Research TransCure, University of Bern, CH-3012 Bern, Switzerland; and cComputational Biomolecular Dynamics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved July 26, 2016 (received for review April 4, 2016) Pathogenic enterobacteria need to survive the extreme acidity of Arg-bound states (10). The two outward-open, substrate-free the stomach to successfully colonize the human gut. Enteric bacteria structures are at the reasonable and moderate resolutions of 3.2 Å circumvent the gastric acid barrier by activating extreme acid- (8) and 3.6 Å (9), respectively, and the only ones available of wild- resistance responses, such as the arginine-dependent acid resistance type AdiC (AdiC-wt). The two other structures are with bound system. In this response, L-arginine is decarboxylated to agmatine, Arg and at 3-Å resolution, and could only be obtained as a result thereby consuming one proton from the cytoplasm. In Escherichia of the introduction of specific point mutations: AdiC-N22A (10) coli,theL-arginine/agmatine antiporter AdiC facilitates the export and AdiC-N101A (11). The N101A mutation results in a defective of agmatine in exchange of L-arginine, thus providing substrates for AdiC protein unable to bind Arg and with a dramatically de- further removal of protons from the cytoplasm and balancing the creased turnover rate compared with wild-type (11).
    [Show full text]
  • Separation of Isomers <Emphasis Type="Italic">L </Emphasis>-Alanine and Sarcosine in Urine by Electrospra
    SHORT COMMUNICATION Separation of Isomers L-Alanine and Sarcosine in Urine by Electrospray Ionization and Tandem Differential Mobility Analysis-Mass Spectrometry Pablo Martínez-Lozanoa and Juan Rusb a Institute for Biomedical Technologies-National Research Council, Milan, Italy b SEADM, Valladolid, Spain Sarcosine, an isomer of L-alanine, has been proposed as a prostate cancer progression biomarker [1]. Both compounds are detected in urine, where the measured sarcosine/alanine ratio has been found to be higher in prostate biopsy-positive group versus controls. We present here preliminary evidence showing that urine samples spiked with sarcosine/alanine can be partially resolved in 3 min via tandem differential mobility analysis-mass spectrometry (DMA-MS). Based on the calibration curves obtained for two mobility peaks, we finally estimate their concentration ratio in urine. (J Am Soc Mass Spectrom 2010, 21, 1129–1132) © 2010 American Society for Mass Spectrometry rostate cancer is a leading cause of death among the tive-ion mode at the DMA entrance slit. Our nanospray male population. Sarcosine, an isomer of alanine, parameters were: capillary 360 ␮m o.d., 50 ␮m i.d., length Pwas recently proposed as a prostate cancer progres- 22 cm, driving pressure 75 mbar, and 3.8 kV. The ions sion biomarker [1]. In particular, the sarcosine/alanine entered the separation region propelled by an electric field ratio was found to be significantly higher in urine derived and against a counterflow gas (0.2 L/min). Sheath gas from biopsy-positive prostate cancer patients compared flow was kept constant, and the classification voltage was with biopsy-negative controls.
    [Show full text]
  • Beta Alanine
    PERFORMANCE ENHANCERS FACTS AND BOTTOM LINE BETA ALANINE What is it? B-alanine is a naturally occurring amino acid (a non-essential amino acid) not used by the body to make muscle tissue. Rather, research has shown that B-alanine works by increasing the muscle content of an important compound – carnosine. In fact, the production of carnosine is limited by the availability of B-alanine. Carnosine is highly concentrated in muscle tissue where its role is primarily to soak up hydrogen ions. Does it work? B-alanine is one of the few dietary supplements that actually have good scientific evidence that it can possibly enhance performance. How does it work? When you exercise intensely the body produces hydrogen ions. The longer you exercise the more hydrogen ions you produce and this reduces the pH level in your muscles. Muscles work best in a very specific pH range and when the pH drops below that level then muscular performance also starts to decrease. Anything that helps to prevent or delay that drop in pH will help delay muscle fatigue. This is where B-alanine has proven to be very helpful. Beta-alanine increases the levels of carnosine in your slow and fast twitch muscle fibers and carnosine is a buffer that basically soaks up hydrogen ions and so reduces the drop in pH. By keeping your hydrogen ion levels lower, B-alanine allows you to train harder and longer. The bottom line is that B-alanine works by increasing the hydrogen ion buffering abilities of your muscles. What benefits does it offer? B-alanine has been shown to increase muscle strength, increase muscle mass, increase anaerobic endurance, increase aerobic endurance and increase exercise capacity.
    [Show full text]
  • Cascade Catalysis–Strategies and Challenges En Route to Preparative
    ChemComm Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/chemcomm Page 1 of 15 ChemComm ChemComm RSCPublishing Feature article Cascade catalysis – strategies and challenges en route to preparative synthetic biology Cite this: DOI: 10.1039/x0xx00000x Jan Muschiol,a,+ Christin Peters,a,+ Nikolin Oberleitner,b Marko D. Mihovilovic,b Uwe T. Bornscheuera and Florian Rudroffb,* Received 00th January 2012, Accepted 00th January 2012 Nature’s smartness and efficiency assembling cascade type reactions inspired biologists and chemists all around the world. Tremendous effort has been put in the understanding and DOI: 10.1039/x0xx00000x mimicking of such networks. In recent years considerable progress has been made in www.rsc.org/ developing multistep one-pot reactions combining either advantage of chemo-, regio-, and stereoselectivity of biocatalysts or promiscuity and productivity of chemocatalysts.
    [Show full text]
  • Poly(L-Alanine) As a Universal Reference Material for Understanding Protein Energies and Structures TERESA HEAD-GORDON, FRANK H
    Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11513-11517, December 1992 Biophysics Poly(L-alanine) as a universal reference material for understanding protein energies and structures TERESA HEAD-GORDON, FRANK H. STILLINGER, MARGARET H. WRIGHT, AND DAVID M. GAY AT&T Bell Laboratories, Murray Hill, NJ 07974 Contributed by Frank H. Stillinger, June 17, 1992 ABSTRACT We present a proposition, the "poly(L- alanine) hypothesis," which asserts that the native backbone geometry for any polypeptide or protein of M residues has a closely mimicking, mechanically stable, image in poly(L- POLY-L-ALANINE alanine) of the same number of residues. Using a molecular co mechanics force field to represent the relevant potential energy a: hypersurfaces, we have carried out calculations over a wide z range of M values to show that poly(L-alanine) possesses the w structural versatility necessary to satisfy the proposition. These w include poly(L-alanine) representatives of minima correspond- U- ing to secondary and supersecondary structures, as well as PROTEIN poly(L-alanine) images for tertiary structures of the naturally occurring proteins bovine pancreatic trypsin inhibitor, crambin, ribonuclease A, and superoxide dismutase. The suc- cessful validation of the hypothesis presented in this paper BACKBONE COORDINATES indicates that poly(L-alanine) will serve as a good reference FIG. 1. Topographic basis ofthe poly(L-alanine) hypothesis. Free material in thermodynamic perturbation theory and calcula- energy hypersurfaces are compared for a given protein and for tions aimed at evaluating relative free energies for competing poly(L-alanine) with the same number of residues. candidate tertiary structures in real polypeptides and proteins.
    [Show full text]
  • Alcohol Use Disorder
    Section: A B C D E Resources References Alcohol Use Disorder (AUD) Tool This tool is designed to support primary care providers (family physicians and primary care nurse practitioners) in screening, diagnosing and implementing pharmacotherapy treatments for adult patients (>18 years) with Alcohol Use Disorder (AUD). Primary care providers should routinely offer medication for moderate and severe AUD. Pharmacotherapy alone to treat AUD is better than no therapy at all.1 Pharmacotherapy is most effective when combined with non-pharmacotherapy, including behavioural therapy, community reinforcement, motivational enhancement, counselling and/or support groups. 2,3 TABLE OF CONTENTS pg. 1 Section A: Screening for AUD pg. 7 Section D: Non-Pharmacotherapy Options pg. 4 Section B: Diagnosing AUD pg. 8 Section E: Alcohol Withdrawal pg. 5 Section C: Pharmacotherapy Options pg. 9 Resources SECTION A: Screening for AUD All patients should be screened routinely (e.g. annually or when indicators are observed) with a recommended tool like the AUDIT. 2,3 It is important to screen all patients and not just patients eliciting an index of suspicion for AUD, since most persons with AUD are not recognized. 4 Consider screening for AUD when any of the following indicators are observed: • After a recent motor vehicle accident • High blood pressure • Liver disease • Frequent work avoidance (off work slips) • Cardiac arrhythmia • Chronic pain • Rosacea • Insomnia • Social problems • Rhinophyma • Exacerbation of sleep apnea • Legal problems Special Patient Populations A few studies have reviewed AUD in specific patient populations, including youth, older adults and pregnant or breastfeeding patients. The AUDIT screening tool considered these populations in determining the sensitivity of the tool.
    [Show full text]
  • Amino Acid Chemistry
    Handout 4 Amino Acid and Protein Chemistry ANSC 619 PHYSIOLOGICAL CHEMISTRY OF LIVESTOCK SPECIES Amino Acid Chemistry I. Chemistry of amino acids A. General amino acid structure + HN3- 1. All amino acids are carboxylic acids, i.e., they have a –COOH group at the #1 carbon. 2. All amino acids contain an amino group at the #2 carbon (may amino acids have a second amino group). 3. All amino acids are zwitterions – they contain both positive and negative charges at physiological pH. II. Essential and nonessential amino acids A. Nonessential amino acids: can make the carbon skeleton 1. From glycolysis. 2. From the TCA cycle. B. Nonessential if it can be made from an essential amino acid. 1. Amino acid "sparing". 2. May still be essential under some conditions. C. Essential amino acids 1. Branched chain amino acids (isoleucine, leucine and valine) 2. Lysine 3. Methionine 4. Phenyalanine 5. Threonine 6. Tryptophan 1 Handout 4 Amino Acid and Protein Chemistry D. Essential during rapid growth or for optimal health 1. Arginine 2. Histidine E. Nonessential amino acids 1. Alanine (from pyruvate) 2. Aspartate, asparagine (from oxaloacetate) 3. Cysteine (from serine and methionine) 4. Glutamate, glutamine (from α-ketoglutarate) 5. Glycine (from serine) 6. Proline (from glutamate) 7. Serine (from 3-phosphoglycerate) 8. Tyrosine (from phenylalanine) E. Nonessential and not required for protein synthesis 1. Hydroxyproline (made postranslationally from proline) 2. Hydroxylysine (made postranslationally from lysine) III. Acidic, basic, polar, and hydrophobic amino acids A. Acidic amino acids: amino acids that can donate a hydrogen ion (proton) and thereby decrease pH in an aqueous solution 1.
    [Show full text]
  • Proline- and Alanine-Rich N-Terminal Extension of the Basic Bovine P-Crystallin B1 Chains
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 161, number 2 FEBS 0790 September 1983 Proline- and alanine-rich N-terminal extension of the basic bovine P-crystallin B1 chains G.A.M. Berbers, W.A. Hoekman, H. Bloemendal, W.W. de Jong, T. Kleinschmidt* and G. Braunitzer* Laboratorium voor Biochemie, Universiteit van Nijmegen, Geert Grooteplein Noord 21, 6525 EZ Nijmegen, The Netherlands and *Max-Planck-Institut ft’ir Biochemie, Abteilung Proteinchemie, D-8033 Martinsried bei Miinchen, FRG Received 22 June 1983 The amino acid sequence of the N-terminal region of the two basic bovine &crystallin B1 chains has been analyzed. The results reveal that @Bibis derived in vivo from the primary gene product @la by removal of a short N-terminal sequence. It appears that them1 chains have the same domain structure as observed in other /3- and y-crystallin chains. They have, however, a very long N-terminal extension in comparison with other &chains. This extension is mainly composed of a remarkable Pro- and Ala-rich sequence, which suggests an interaction of these structural proteins with the cytoskeleton and/or the plasma membranes of the lens cells. Protein sequence Bovine &crystallin N-terminal extension Proline- and aianine-rich Domain structure 1. INTRODUCTION 33 000 and 31000, respectively, are characteristic for fltikt, [ 111. ,8Fha is a primary gene product, from The crystallins are evolutionary highly conserv- which ,8&b arises by post-translational modifica- ed structural eye lens proteins, which can be divid; tion [lo-121, most probably a proteolytic step ed into 4 classes: (Y-,,&, y- and t-crystallin [ 11.
    [Show full text]
  • The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats
    W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 4-2017 The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats Justin P. Canakis College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Animal Sciences Commons, Exercise Science Commons, Laboratory and Basic Science Research Commons, and the Other Nutrition Commons Recommended Citation Canakis, Justin P., "The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats" (2017). Undergraduate Honors Theses. Paper 1128. https://scholarworks.wm.edu/honorstheses/1128 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. 1 2 Title Page……………………………………………………………………………………...…..1 Abstract…………………………………………………………………………………………....5 Acknowledgement……………………………………………………………………….………..6 Background.………………………………………………………………………..……………...7 DSEHA and its Effect on the VMS Industry………………...……………………………7 History of Adverse Side Effects from Pre-Workout Supplements ………….……………8 Ingredient Analysis ………………………………………..……………….……………..……....9 2.5g Beta-Alanine…………………………..……………………………………………..9 1g Creatine Nitrate……………………………...……………………………………..…12 500mg L-Leucine……………………………………………………………………...…16 500mg Agmatine Sulfate……………………….………………………………..………18
    [Show full text]
  • The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by College of William & Mary: W&M Publish W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 4-2017 The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats Justin P. Canakis College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Animal Sciences Commons, Exercise Science Commons, Laboratory and Basic Science Research Commons, and the Other Nutrition Commons Recommended Citation Canakis, Justin P., "The Efficacy and Safety of Six-Weeks of Pre-Workout Supplementation in Resistance Trained Rats" (2017). Undergraduate Honors Theses. Paper 1128. https://scholarworks.wm.edu/honorstheses/1128 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. 1 2 Title Page……………………………………………………………………………………...…..1 Abstract…………………………………………………………………………………………....5 Acknowledgement……………………………………………………………………….………..6 Background.………………………………………………………………………..……………...7 DSEHA and its Effect on the VMS Industry………………...……………………………7 History of Adverse Side Effects from Pre-Workout Supplements ………….……………8 Ingredient Analysis ………………………………………..……………….……………..……....9 2.5g Beta-Alanine…………………………..……………………………………………..9
    [Show full text]
  • Table of Contents
    Development of Transaminases for the Synthesis of Enantiomerically Pure Chiral Amines A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences. 2012 Jennifer Hopwood School of Chemistry Table of contents Table of contents ............................................................................................................. 4 Abstract ............................................................................................................................ 9 Declaration ..................................................................................................................... 10 Copyright statement...................................................................................................... 10 Acknowledgements ........................................................................................................ 11 Abbreviations ................................................................................................................ 12 1. Introduction ........................................................................................................... 14 1.1 Biocatalysis ...................................................................................................... 14 1.2 Enzymatic synthesis of chiral amines .............................................................. 18 1.2.1 Chiral amines ............................................................................................ 18 1.2.2 Biocatalytic
    [Show full text]
  • Purification and Characterization of Alanine Dehydrogenase from a Cyanobacterium, Phormidium Lapideum
    J. Biochem. 116, 995-1000 (1994) Purification and Characterization of Alanine Dehydrogenase from a Cyanobacterium, Phormidium lapideum Yoshihiro Sawa,1 Masaaki Tani, Ken Murata, Hitoshi Shibata, and Hideo Ochiai Department of Applied Biochemistry, Faculty of Agriculture, Shimane University, Matsue, Shimane 690 Received for publication, May 12, 1994 Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0mM for L-alanine and 0.04mM for NAD+, 0.33mM for pyruvate, 60.6mM for NH4+ (pH 8.7), and 0.02mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5•L-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP. Key words: alanine dehydrogenase, cyanobacterium, Phormidium lapideum. Alanine dehydrogenase [L-alanine: NAD+ oxidoreductase, enzymological and regulatory aspects of AlaDH from deaminating, EC 1.4.1.1] catalyzes a reversible oxidative cyanobacteria, we have purified the enzyme from a non deamination of L-alanine to pyruvate and is found in -N2-fixing thermophilic cyanobacterium, Phormidium lapi Bacillus species and some other bacteria (1, 2).
    [Show full text]