DOCSLIB.ORG

Amino Acid Side Chain Conformation in Angiotensin II and Analogs

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Amino Acid Side Chain Conformation in Angiotensin II and Analogs Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, pp. 82-86, January 1980 Biochemistry Amino acid side chain conformation in angiotensin II and analogs: Correlated results of circular dichroism and 1H nuclear magnetic resonance (peptide hormones/competitive inhibitor/N-methylation/pH titration) F. PIRIou*, K. LINTNER*, S. FERMANDJIAN*, P. FROMAGEOT*, M. C. KHOSLAt, R. R. SMEBYt, AND F. M. BUMPUSt *Service de Biochimie, D)partement de Biologie, Centre d'Etudes Nucl&aires de Saclay, P.B. No. 2, F-91190 Gif-sur-Yvette, France; and tThe Clinic Center, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44106 Communicated by Irvine H. Page, September 4,1979 ABSTRACT [1-Sarcosine,S-isoleucinelangiotensin II (Sar- [Sarl,11e8]Angiotensin II was shown to be a potent antagonist Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent an- of the pressor action of antiotensin II (Asp-Arg-Val-Tyr-Ile- tagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue His-Pro-Phe). However, this and other similar antagonistic at position 4 (tyrosine) or position 5 (isoleucine) was replaced peptides have short half-lives in vivo; the shortness is presum- with the corresponding N-methylated residue. This change ably due to rapid degradation by peptidases (for detailed re- drastically reduced the antagonistic properties of this analog. views, see refs. 1 and 2). With a view to render these peptides The present work was therefore undertaken to investigate the more resistant to enzymatic degradation, the residue at position effect of N-methylation on overall conformation of these pep- tides and to determine the conformational requirements for 4 (tyrosine) or position 5 (isoleucine) was replaced with the maximum agonistic or antagonistic pro rties. Conformation corresponding N-methylated amino acid. The analogs thus studies were carried out by circular dichroism and proton nu- synthesized, [Sar',MeTyr4,I1e81- and [Sar1,MeIle5,Ile8jangio- clear magnetic resonance spectroscopy in aqueous solution as tensin II showed drastically reduced antagonistic properties (3, a function of pH. The results indicated that: (i) angiotensin II to N-methylation and [1-sarcosine,8-isoleucinelangiotensin II gave practically 4). It is possible that the reduced activity due identical spectroscopic data; and (ii) N-methylation in either may be a consequence of either modification of backbone position 4 or position 5 resulted in remarkable changes in the conformation or rotational restriction of the side chain, or both. peptide backbone and a severe limitation in rotational freedom It has been shown that N-methylation of a single amino acid of side chains in tyrosine, isoleucine, and histidine residues. residue in a peptide chain restricts the number of conformations However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyro- not only of the N-methylated residues but also of the residue sine,S-isoleucinelangiotensin II than in [1-sarcosine,5-N- preceding it (5, 6). Further, N-methylation may also affect the methylisoleucine,S-isoleucinejlangiotensin IL Thus, these results lowest energy conformation of the involved fragment. There- suggest that: (I) the backbone and side chain structure of a potent fore, the conformations of the above analogs and those of an- angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing giotensin II and [Sar',Ile8Jangiotensin II in water solution were and binding with the receptor on the cell membrane; and (ii) examined as a function of pH. Circular dichroism (CD) and greater impact of N-methylation in position 5 on the overall proton NMR spectroscopy were used to obtain information conformation of these peptides points to the controlling influ- about the side-chain conformations, especially with regard to ence of position 5 (isoleucine) in aligning the residues in the those residues that are important for biological activity, namely, central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly ex- tyrosine and histidine. tended structure. Any change in this arrangement may lead to reduced biological activity. MATERIALS AND METHODS The synthesis and purification of the peptides have been de- Recognition of a biologically active molecule by its receptor site scribed (3, 4). CD spectra were recorded on a Dichrograph is probably the first event of the hormone-receptor interaction concentrations were process, whereas binding and signal release are subsequent model Mark III (Jobin Yvon). Peptide phenomena. Thus, it is likely that during the approach of a made to about 0.5 mM and were determined by ultraviolet peptide to the cell membrane the local environment may absorbance (E275 = 1350 M-' cm-' at pH 5.8). The previously greatly influence the selection of a "recognizable" conformation described method of pH titration was used without modifica- among numerous other conformations. It is therefore assumed tion (7). Theoretical titration curves were calculated with the that the peptides showing physical properties similar to those help of a computer program "Titrage" kindly made available of the parent hormone, in a given set of experimental conditions by M. Gingold. Results are expressed in molar ellipticity [0I = (e.g., solvent and pH effects, etc.), may survive the conforma- 3300 Ae, in units of ocm2/dmol. tional selection procedure during their approach to the cell 'H NMR spectra were recorded at 250 MHz on a CAMECA membrane. The present work was undertaken to provide evi- TSN 250 instrument in the Fourier transform mode. The dence for this hypothesis, by using angiotensin analogues as peptide concentration in 2H20 was 35 mM. The internal ref- model peptides. erence was sodium 2,2,3,3-tetradeutero-3-trimethylsilylpro- pionate; the lock was on 2H; and the temperature was 220C (except for the ca protons of histidine in [Sarl,Ile8langiotensin The publication costs of this article were defrayed in part by page was to shift the HO2H charge payment. This article must therefore be hereby marked "ad- II, for which 40'C necessary peak). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: CD, circular dichroism. 82 Downloaded by guest on September 25, 2021 Biochemistry: Piriou et al. Proc. Natl. Acad. Sci. USA 77 (1980) 83 RESULTS Histidine Titration. Arguments based on data from a large CD number of analogs studied (9) have shown that the inversion Marked differences were observed in the CD spectra of the four of the tyrosine signal in angiotensin II (Fig. lB) is due to the peptides dissolved in water at pH 5.8, recorded in the aromatic titration of the close-up histidine side chain. The amplitude of region (320-250 nm). The sign and the intensity of the tyrosine the titratiop curve observed for a given peptide is a function of signal at the initial pH value (5.8) are given in Fig. 1, in which its conformational characteristics especially with respect to the the values for [Sar',MeIle5,Ile8]angiotensin II (+450) and central residues 3 to 7 of angiotensin 11 (8). The curves in Fig. [Sarl,MeTyr4,Ile8langiotensin II (-850) are noteworthy. Signal 1B follow this pattern. Angiotensin II and its potent inhibitor, intensity, which in tyrosine model compounds and previously [Sarl,Ile8]angiotensin II, have curves that are almost superim- studied angiotensin II analogs was found to be in the +300 posable (A[0J = -450), which confirms the titration behavior range, is of limited diagnostic value and indicates at best the of the less-modified inhibitor [Ile8]angiotensin II (8). A marked amount of local asymmetry or hindered rotational freedom of reduction in the side-chain-side-chain influence is observed the chromophore. On the other hand, the comparison of titra- for [Sar',MeTyr4;Ile8]angiotensin II (A[O] = -200). The in- tion curves obtained for different peptides yields precise tensity of the 'Lb band in this peptide hints at hindered rotation structural information (8). This is also true for the peptides of the tyrosine side chain, which could account for an increased presented here, for which we shall discuss the three zones of pH time-averaged distance between the two aromatic side chains shown in Fig. 1. tyrosine and histidine. Again, these factors are possibly tied in Carboxyl Titration. The intensity of the 'Lb signal of tyrosine with as yet unquantifiable changes in backbone conformation. in angiotensin II is sensitive to the titration of one or more The titration curve for [Sarl,MeIle5,le8]angiotensin II, on the carboxyl groups (Fig. 1A) (8). It had been observed that the other hand, is flat in this pH region; the respective orientation slope of ihe titration curve is dependent on the presence or of the tyrosine arnd histidine side chains and the distance be- absence of the f3-carboxyl group in position 1: it is positive in tween them has clearly been upset by the N-methylation and [Aspl]angiotensin II peptides but negative in [Asn']angiotensin the concomitant changes of dihedral angles 4 and i/' along the II, [Me2Glyl]angiotensin II, and des-Aspl-angiotensin II, etc. central stretch of the backbone. (8). Whereas the possibility of conformational changes in the Phenol Titratidn. It is only between pH 9 and pH 12 that we backbone structure during COOH titration or an indirect titrate the chromophore of the tyrosine side-chain proper. The effect via the histidine side chain cannot be denied, the relative change in electronic structure causes the well-documented shift distance and orientation of the carboxyl group toward the ty- of the 'Lb band from 275 nm to 293 nm; in almost all cases so rosine side chain is likely to be the main factor leading to the far published in the literature or observed in our laboratory observed effects.
Load more

Recommended publications
  • Effect of Peptide Histidine Isoleucine on Water and Electrolyte Transport in the Human Jejunum
    Gut: first published as 10.1136/gut.25.6.624 on 1 June 1984. Downloaded from Gut, 1984, 25, 624-628 Alimentary tract and pancreas Effect of peptide histidine isoleucine on water and electrolyte transport in the human jejunum K J MORIARTY, J E HEGARTY, K TATEMOTO, V MUTT, N D CHRISTOFIDES, S R BLOOM, AND J R WOOD From the Department of Gastroenterology, St Bartholomew's Hospital, London, The Liver Unit, King's College Hospital, London, Department ofMedicine, Hammersmith Hospital, London, and Department of Biochemistry, Karolinska Institute, Stockholm, Sweden SUMMARY Peptide histidine isoleucine, a 27 amino acid peptide with close amino acid sequence homology to vasoactive intestinal peptide and secretin, is distributed throughout the mammalian intestinal tract, where it has been localised to intramural neurones. An intestinal perfusion technique has been used to study the effect of intravenous peptide histidine isoleucine (44.5 pmol/kg/min) on water and electrolyte transport from a plasma like electrolyte solution in human jejunum in vivo. Peptide histidine isoleucine infusion produced peak plasma peptide histidine isoleucine concentrations in the range 2000-3000 pmolIl, flushing, tachycardia and a reduction in diastolic blood pressure. Peptide histidine isoleucine caused a significant inhibition of net absorption of water, sodium, potassium and bicarbonate and induced a net secretion of chloride, these changes being completely reversed during the post-peptide histidine isoleucine period. These findings suggest that endogenous peptide histidine isoleucine may participate in the neurohumoral regulation of water and electrolyte transport in the human jejunum. http://gut.bmj.com/ Peptide histidine isoleucine, isolated originally from INTESTINAL PERFUSION mammalian small intestine, is a 27-amino acid After an eight hour fast, each subject swallowed a peptide having close amino acid sequence homology double lumen intestinal perfusion tube, incorpo- to vasoactive intestinal peptide and secretin.
    [Show full text]
  • Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis Kazumoto Murata1 and Masami Moriyama2
    Research Article Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis Kazumoto Murata1 and Masami Moriyama2 1The First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan and 2Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan Abstract infection in the airways of cystic fibrosis (8), and susceptibility to salmonella infection in mouse intestinal tracts (9). In addition to In spite of recent advances in the treatment of colon cancer, h multiple liver metastases of colon cancer are still difficult to their direct antimicrobial activities, -defensins are strong treat. Some chemotherapeutic regimens have been reported to chemotactic factors for memory T cells and dendritic cells, be efficient, but there is a high risk of side effects associated suggesting that they also play an important role in acquired immunity (10–12). h-defensins are also inducible by inflammatory with these. Here, we show that isoleucine, an essential amino a h acid, prevents liver metastases in a mouse colon cancer cytokines, such as tumor necrosis factor- and interleukin-1 (13, 14). Recently, Fehlbaum et al. (15) reported that isoleucine metastatic model. Because isoleucine is a strong inducer of h B-defensin, we first hypothesized that it prevented liver and its analogues are highly specific inducers of -defensins. We thus originally hypothesized that isoleucine may contribute to metastases via the accumulation of dendritic cells or memory B tumor immunity through both innate and acquired immunity by T cells through up-regulation of -defensin. However, neither h B-defensin nor immunologic responses were induced by induction of -defensins.
    [Show full text]
  • Workshop 1 – Biochemistry (Chem 160)
    Workshop 1 – Biochemistry (Chem 160) 1. Draw the following peptide at pH = 7 and make sure to include the overall charge, label the N- and C-terminus, the peptide bond and the -carbon. AVDKY Give the overall charge of the peptide at pH = 3 and 12. 2. Draw a titration curve for Arg, make sure to label the different points. Determine the pI for Arg. 3. Nonpolar solute + water = solution a. What is the S of the universe, system and surroundings? The S of the universe would decrease this is why it is not spontaneous, the S of the system would increase but to a lesser extent to which the S of the surrounding would decrease S universe = S system + S surroundings 4. What is the hydrophobic effect and explain why it is thermodynamically favorable. The hydrophobic effect is when hydrophobic molecules tend to clump together burying them and placing hydrophilic molecules on the outside. The reason this is thermodynamically favorable is because it frees caged water molecules when burying clumping the hydrophobic molecules together. 5. Urea dissolves very readily in water, but the solution becomes very cold as the urea dissolves. How is this possible? Urea dissolves in water because when dissolving there is a net increase in entropy of the universe. The heat exchange, getting colder only reflects the enthalpy (H) component of the total energy change. The entropy change is high enough to offset the enthalpy component and to add up to an overall -G 6. A mutation that changes an alanine residue in the interior of a protein to valine is found to lead to a loss of activity.
    [Show full text]
  • The Optimum Isoleucine:Lysine Ratio in Starter Diets to Maximize Growth Performance of the Early-Weaned Pig
    Kansas Agricultural Experiment Station Research Reports Volume 0 Issue 10 Swine Day (1968-2014) Article 837 2000 The optimum isoleucine:lysine ratio in starter diets to maximize growth performance of the early-weaned pig B W. James Robert D. Goodband Michael D. Tokach See next page for additional authors Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Other Animal Sciences Commons Recommended Citation James, B W.; Goodband, Robert D.; Tokach, Michael D.; Nelssen, Jim L.; and DeRouchey, Joel M. (2000) "The optimum isoleucine:lysine ratio in starter diets to maximize growth performance of the early-weaned pig," Kansas Agricultural Experiment Station Research Reports: Vol. 0: Iss. 10. https://doi.org/10.4148/ 2378-5977.6677 This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2000 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. The optimum isoleucine:lysine ratio in starter diets to maximize growth performance of the early-weaned pig Abstract A total of 360 weanling pigs (initially 12.3 lb BW and approximately 18 d of age) was used in a 14-d growth assay to determine the optimal isoleucine:lysine ratio to maximize growth performance.
    [Show full text]
  • ACE2–Angiotensin-(1–7)–Mas Axis and Oxidative Stress in Cardiovascular Disease
    Hypertension Research (2011) 34, 154–160 & 2011 The Japanese Society of Hypertension All rights reserved 0916-9636/11 $32.00 www.nature.com/hr REVIEW SERIES ACE2–angiotensin-(1–7)–Mas axis and oxidative stress in cardiovascular disease Luiza A Rabelo1,2, Natalia Alenina1 and Michael Bader1 The renin–angiotensin–aldosterone system (RAAS) is a pivotal regulator of physiological homeostasis and diseases of the cardiovascular system. Recently, new factors have been discovered, such as angiotensin-converting enzyme 2 (ACE2), angiotensin-(1–7) and Mas. This newly defined ACE2–angiotensin-(1–7)–Mas axis was shown to have a critical role in the vasculature and in the heart, exerting mainly protective effects. One important mechanism of the classic and the new RAAS regulate vascular function is through the regulation of redox signaling. Angiotensin II is a classic prooxidant peptide that increases superoxide production through the activation of NAD(P)H oxidases. This review summarizes the current knowledge about the ACE2–angiotensin-(1–7)–Mas axis and redox signaling in the context of cardiovascular regulation and disease. By interacting with its receptor Mas, angiotensin-(1–7) induces the release of nitric oxide from endothelial cells and thereby counteracts the effects of angiotensin II. ACE2 converts angiotensin II to angiotensin-(1–7) and, thus, is a pivotal regulator of the local effects of the RAAS on the vessel wall. Taken together, the ACE2–angiotensin-(1–7)–Mas axis emerges as a novel therapeutic target in the context of cardiovascular
    [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]
  • Amino Acids Amino Acids
    Amino Acids Amino Acids What Are Amino Acids? Essential Amino Acids Non Essential Amino Acids Amino acids are the building blocks of proteins; proteins are made of amino acids. Isoleucine Arginine (conditional) When you ingest a protein your body breaks it down into the individual aminos, Leucine Glutamine (conditional) reorders them, re-folds them, and turns them into whatever is needed by the body at Lysine Tyrosine (conditional) that time. From only 20 amino acids, the body is able to make thousands of unique proteins with different functions. Methionine Cysteine (conditional) Phenylalanine Glycine (conditional) Threonine Proline (conditional) Did You Know? Tryptophan Serine (conditional) Valine Ornithine (conditional) There are 20 different types of amino acids that can be combined to make a protein. Each protein consists of 50 to 2,000 amino acids that are connected together in a specific Histidine* Alanine sequence. The sequence of the amino acids determines each protein’s unique structure Asparagine and its specific function in the body. Asparate Popular Amino Acid Supplements How Do They Benefit Our Health? Acetyl L- Carnitine: As part of its role in supporting L-Lysine: L-Lysine, an essential amino acid, is mental function, Acetyl L-Carnitine may help needed to support proper growth and bone Proteins (amino acids) are needed by your body to maintain muscles, bones, blood, as support memory, attention span and mental development. It can also support immune function. well as create enzymes, neurotransmitters and antibodies, as well as transport and performance. store molecules. N-Acetyl Cysteine: N-Acetyl Cysteine (NAC) is a L-Arginine: L-Arginine is a nonessential amino acid form of the amino acid cysteine.
    [Show full text]
  • Solutions to 7.012 Problem Set 1
    MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Solutions to 7.012 Problem Set 1 Question 1 Bob, a student taking 7.012, looks at a long-standing puddle outside his dorm window. Curious as to what was growing in the cloudy water, he takes a sample to his TA, Brad Student. He wanted to know whether the organisms in the sample were prokaryotic or eukaryotic. a) Give an example of a prokaryotic and a eukaryotic organism. Prokaryotic: Eukaryotic: All bacteria Yeast, fungi, any animial or plant b) Using a light microscope, how could he tell the difference between a prokaryotic organism and a eukaryotic one? The resolution of the light microscope would allow you to see if the cell had a true nucleus or organelles. A cell with a true nucleus and organelles would be eukaryotic. You could also determine size, but that may not be sufficient to establish whether a cell is prokaryotic or eukaryotic. c) What additional differences exist between prokaryotic and eukaryotic organisms? Any answer from above also fine here. In addition, prokaryotic and eukaryotic organisms differ at the DNA level. Eukaryotes have more complex genomes than prokaryotes do. Question 2 A new startup company hires you to help with their product development. Your task is to find a protein that interacts with a polysaccharide. a) You find a large protein that has a single binding site for the polysaccharide cellulose. Which amino acids might you expect to find in the binding pocket of the protein? What is the strongest type of interaction possible between these amino acids and the cellulose? Cellulose is a polymer of glucose and as such has many free hydroxyl groups.
    [Show full text]
  • Valine and Isoleucine: the Next Limiting Amino Acids in Broiler Diets Vol
    Valine and Isoleucine: The next limiting amino acids in broiler diets Vol. 46 (1), April 2011, Page 59 Valine and Isoleucine: The next limiting amino acids in broiler diets Etienne Corrent (Ajinomoto Eurolysine S.A.S.) and Dr. Jörg Bartelt (Lohmann Animal Health GmbH & Co. KG) Drastic genetic changes have occurred in many commercial broiler lines during the last years with regard to performance. This genetic improvement needs a corresponding adjustment of our knowledge about amino acid nutrition in broilers. Additionally, today broiler feed formulators are not only focused on minimising the costs. They also have to take into consideration environmental issues and the impact of feed on broiler health. Reducing excess dietary crude protein (CP) is an important way of addressing these issues. The least cost formulation of the diet according to the ideal protein concept is the best way to supply an economic and a balanced amino acids feed for broiler, which can help to reduce the nitrogen excretion during the rearing period. What are the next limiting amino acids in broiler diets? To reduce dietary crude protein levels in broiler feed, it is necessary to know which indispensable amino acids become limiting in diets and what the requirement of broilers is. The usage of feed use amino acids (methionine sources, L-Lysine sources, L-Threonine) in broiler feed is well established. Depending on the requirement assumed for each amino acid, Valine, Isoleucine, Tryptophan and Arginine are generally considered as the next limiting amino acids in broiler feed. Indeed, the amino acid composition of protein differs between feedstuffs and can impact the order in which amino acids become limiting in diets.
    [Show full text]
  • Inhibition of Glycine Oxidation in Cultured Fibroblasts by Isoleucine
    Pediat. Res. 7: 945-947 (1973) Glycine isoleucine hyperglycemia Inhibition of Glycine Oxidation in Cultured Fibroblasts by Isoleucine RICHARD E. HILLMAN, [1S1 LUCILLE H. SOWERS, AND JACK L. COHEN Division of Medical Genetics, Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri USA Extract Cultured fibroblasts were shown to oxidize glycine to CO2- Isoleucine (10 HIM) in- hibited glycine oxidation to CO2 by about 60% in a concentration range of from 0.025 to 10 mM glycine in fibroblasts grown from a patient with ^-ketothiolase defi- cienty. Glycine oxidation by control cell lines was not inhibited by isoleucine. These studies demonstrate an interrelation between isoleucine catabolism and glycine oxida- tion in fibroblasts cultured from a patient with the ketotic hyperglycinemia syndrome. Speculation Hyperglycinemia and hyperglycinuria seen in the "ketotic hyperglycinemia" syn- drome would appear to be secondary to accumulation of products of isoleucine catabolism. Thus, the varying levels of glycine reported in the serum and urine of these patients probably reflect differences in protein and isoleucine intake than rather primary blocks in glycine metabolism. Introduction deficiency [6], methylmalonyl-CoA mutase deficiency [8], and /?-ketothiolase deficiency [3]. a-Methyl-/3-hy- Since its original description by Childs et at. [2], the droxybutyrate was found in the patient with /?-keto- "ketotic hyperglycinemia" syndrome has been shown thiolase deficiency [3]. to be associated with three different defects in the pathway from isoleucine to succinyl-CoA. The sister of There has never been a satisfactory explanation of Childs' original patient was demonstrated to have pro- the elevated levels of glycine in serum and urine of pionyl-CoA carboxylase deficiency [4], other cases have patients with these disorders of isoleucine metabolism.
    [Show full text]
  • Amino Acid Requirements of the Free-Living Nematode Caenorhabditis Briggsae
    AMINO ACID REQUIREMENTS OF THE FREE-LIVING NEMATODE CAENORHABDITIS BRIGGSAE BY J. R. VANFLETEREN Instituut voor Dierkunde, Laboratoria voor Morfologie en Systematiek, RijksuniversiteitGent, Belgium Washed yeast ribosomes promote growth and reproduction of C. briggsae, even when supple- mented to the basal medium at dosages too low to provide the organisms with sufficient amounts of essential amino acids. Hence, a re-investigation of the amino acid requirements of C. briggsae by single and multiple omission of amino acids from the basal medium revealed unambiguously that arginine, histidine, lysine, tryptophan, phenylalanine, methionine, threonine, leucine, isoleucine and valine are not synthetized at levels to permit reproduction; they are called essential amino acids. The requirement for arginine and isoleucinehowever appears to be less clear-cut. On the contrary, evidence is presented that alanine, asparagine, cysteine, glutamate, glutamine, glycine, proline, serine and tryosine can be synthetized at adequate levels; they are called non- essential amino acids. In addition it was shown that multiple omission of the non-essential amino acids is not deleterious. This is believed to be an important step towards the development of a minimum essential medium (MEM) for growth and reproduction of C. briggsae. Sustained growth of the free-living nematode Caenorhabditis brigg.rae can be obtained on a chemically defined medium, supplemented with adequate levels of a proteinaceous growth factor. The most satisfactory, chemically defined medium hitherto reported (Buecher, Hansen & Yarwood, 1966), has been called C. brigg.iae Maintenance Medium (CbMM) and is now commercially available. CbMM is an extremely rich medium, being composed of 53 components, all present at high concentrations.
    [Show full text]
  • Recent Progress in the Microbial Production of Pyruvic Acid
    fermentation Review Recent Progress in the Microbial Production of Pyruvic Acid Neda Maleki 1 and Mark A. Eiteman 2,* 1 Department of Food Science, Engineering and Technology, University of Tehran, Karaj 31587-77871, Iran; [email protected] 2 School of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, GA 30602, USA * Correspondence: [email protected]; Tel.: +1-706-542-0833 Academic Editor: Gunnar Lidén Received: 10 January 2017; Accepted: 6 February 2017; Published: 13 February 2017 Abstract: Pyruvic acid (pyruvate) is a cellular metabolite found at the biochemical junction of glycolysis and the tricarboxylic acid cycle. Pyruvate is used in food, cosmetics, pharmaceutical and agricultural applications. Microbial production of pyruvate from either yeast or bacteria relies on restricting the natural catabolism of pyruvate, while also limiting the accumulation of the numerous potential by-products. In this review we describe research to improve pyruvate formation which has targeted both strain development and process development. Strain development requires an understanding of carbohydrate metabolism and the many competing enzymes which use pyruvate as a substrate, and it often combines classical mutation/isolation approaches with modern metabolic engineering strategies. Process development requires an understanding of operational modes and their differing effects on microbial growth and product formation. Keywords: auxotrophy; Candida glabrata; Escherichia coli; fed-batch; metabolic engineering; pyruvate; pyruvate dehydrogenase 1. Introduction Pyruvic acid (pyruvate at neutral pH) is a three carbon oxo-monocarboxylic acid, also known as 2-oxopropanoic acid, 2-ketopropionic acid or acetylformic acid. Pyruvate is biochemically located at the end of glycolysis and entry into the tricarboxylic acid (TCA) cycle (Figure1).
    [Show full text]