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Controlled Biosynthesis of Actinomycin with Sarcosine

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458 J. F. BERRY AND V. P. WHITTAKER I959 Henschler, D. (1956). Hoppe-Seyl. Z. 305, 97. Melchior, N. C. & Melchior, J. B. (1956). Science, 124, 402. Henschler, D. (1957). Hoppe-Seyl. Z. 309, 276. Mounter, L. A. (1952). Biochem. J. 50, 122. Hestrin, S. (1949). J. biol. Chem. 180, 249. Nachmansohn, D. & John, H. M. (1945). J. biol. Chem. 158, Hestrin, S. (1950). Biochim. biophys. Acta, 4, 310. 157. Holtz, P. & Schumann, H. G. (1954). Naturwissen8chaften, Neuberger, A. (1938). Biochem. J. 32, 1452. 41, 306. Noda, L. H., Kuby, S. A. & Lardy, H. A. (1953). J. Amer. Kalckar, H. M. (1947). J. biol. Chem. 167, 461. chem. Soc. 75, 913. Kaplan, N. 0. & Lipmann, F. (1948). J. biol. Chem. 174,37. Raw, I. (1953). Science, 118, 159. Kennedy, E. P. (1956). Canad. J. Biochem. Physiol. 34, Reid, R. L. & Lederer, M. (1951). Biochem. J. 50, 60. 334. Smallman, B. N. (1958). J. Neurochem. 2, 119. Keyl, M. J., Michaelson, I. A. & Whittaker, V. P. (1957). Stadtman, E. R. (1952). J. biol. Chem. 198, 535. J. Physiol. 139, 434. Stadtman, E. R. (1953). J. cell. comp. Physiol. 41, Suppl. 1, Kielley, W. W., Stadtman, E. R. & Bradley, L. B. (1954). 89. In Glutathione: A Symposium, p. 57. Ed. by Colowick, Stadtman, E. R. & White, F. H. (1953). J. Amer. chem. S. P. et al. New York: Academic Press Inc. Soc. 75, 2022. Korey, S. R., de Braganza, B. & Nachmansohn, D. (1951). Strecker, H. J., Mela, P. & Waelsch, H. (1955). J. biol. J. biol. Chem. 189, 705.. Chem. 212, 223. Korff, R. W. von (1953). J. biol. Chem. 203, 265. Torda, C. & Wolff, H. G. (1945). Proc. Soc. exp. Biol., N. Y., Kumagai, H. & Ebashi, S. (1954). Nature, Lond., 173, 871. 59, 246. Lipmann, F. & Tuttle, L. C. (1945a). J. biol. Chem. 159, 21. Vignais, P. M., Gallagher, C. H. & Zabin, I. (1958). Lipmann, F. & Tuttle, L. C. (1945b). J. biol. Chem. 161, J. Neurochem. 2, 283. 415. Whittaker, V. P. (1953). Unpublished results quoted in Lipmann, F. & Tuttle, L. C. (1950). Biochim. biophys. Acta, Lectures on the Scientific Basi8 of Medicine, vol. 6, p. 198. 4, 301. Ed. by Fraser, F. R. London: Athlone Press. Mahler, H. R., Wakil, S. J. & Bock, R. M. (1953). J. biol. Whittaker, V. P. & Wijesundera, S. (1952). Biochem. J. 51, Chem. 204, 453. 348. Controlled Biosynthesis of Actinomycin with Sarcosine BY E. KATZ AND W. A. GOSS In8titute of Microbiology, Rutger8, The State Univer8ity, New Bru?nwick, New Jer8ey, U.S.A. (Received 3 April 1959) The actinomycins represent a family of chromo- New actinomycins were formed by S. chryaomallus peptide antibiotics which differ solely in the nature when DL-isoleucine or sarcosine was added to the of the amino acids present in the peptides of the medium (Schmidt-Kastner, 1956a). In our Lab- molecule (Fig. 1). It has been established that an oratory it has been determined that S. antiobiticus actinomycin-producing organism generally syn- forms at least one new actinomycin with sarcosine thesizes a mixture of these substances. For and several new compounds when DL-pipecolic acid example, Streptomyces antibioticu8 forms a mixture is used. consisting of actinomycins I-V; occasionally trace During an investigation of the role of amino amounts of a sixth component are produced acids on actinomycin synthesis by S. antibioticus, it (Katz & Goss, 1958). Streptomyces chry8omallu8 was observed that sarcosine selectively stimulated produces actinomycins IV, VI and VII (Schmidt- the production of actinomycins II and III (Fig. 2). Kastner, 1956b). These components, normally synthesized in trace The quantitative and qualitative nature of the amounts, represented approximately 60 % of the actinomycin mixture synthesized can be modified actinomycin mixture formed under the nutritional to a considerable extent; in particular, the nitrogen conditions employed. By paper-chromatography source supplied has been shown to have a profound techniques, it was established that actinomycin II influence on its composition. Actinomycin IV in- contains threonine, valine, sarcosine, and N- creased from 10 to 83 % ofthe mixture produced by methylvaline, whereas actinomycin III possesses, S. chry8omallu8 when DL-valine was added to the in addition, one-half the amount ofproline found in medium (Schmidt-Kastner, 1956b); hydroxy-L- actinomycin IV (Katz, Goss & Pugh, 1958). proline brought about an increase in synthesis of Recently Johnson & Mauger (1959) obtained actinomycin I from 6-7 to 31 % of the actinomycins quantitative data showing that 4 moles of sarcosine produced by S. antibioticu, (Katz & Goss, 1958). and no proline are present in actinomycin II, and VoI. 73 CONTROLLED BIOSYNTHESIS OF ACTINOMYCIN 459 that 3 moles of sarcosine and only 1 mole of proline Chemical Co., Norwich, N.Y., U.S.A.), 25 g., Bacto beef are in actinomycin III. They proposed for actino- extract 10 g., tap water 11., pH 7 0, was employed for mycins II and III the structures indicated in the growth of the organism. legend of Fig. 1. Production medium. A chemically defined medium con- taining L-glutamic acid 2-0 g., D-galactose 10.0 g., K2HPO4 Schmidt-Kastner (1956b) has suggested that 1.0 g., MgSO4,7H20 0-025 g., CaCl2,2H20 0-025 g., ZnSO4, exogenous sarcosine interfered with the incorpora- 7H2O 0-025 g., FeSO4,7H20 0-025 g., distilled water 1 1., tion of proline into certain actinomycin peptides. pH 7-2-7-3, was used for actinomycin production. Amounts The results obtained in our studies provide evidence (100 ml.) of medium were distributed into 250 ml. Erlen- for the view that sarcosine competes with proline meyer flasks and sterilized at 15 lb./in.2 pressure and 1210 and replaces it in certain actinomycin peptides. for 15 min. D-Galactose was autoclaved separately in a similar manner and added to the medium just before in- oculation. Production of actinomycin was carried out in EXPERIMENTAL shaken cultures (240 rev./min.) at 280. Aqueous solutions Culture. Streptomyces antibioticu8, strain 3720, was used of sarcosine and other amino acids generally were added at throughout the investigation. The procedure for prepara- the onset of actinomycin synthesis (1-3 days after inocula- tion of an inoculum has been described previously (Goss & tion), unless specified otherwise. Katz, 1957). An N-Z amine medium consisting of N-Z Determination of actinomycin potency. The antibiotic amine A (a pancreatic digest of casein prepared by Sheffield titre of culture filtrates was determined by a paper-disk method of bioassay (Goss & Katz, 1957). H3C CH3 H3C H3 fH fH CO CH (L) (L) CH CO N-CR3 N-CH3 Sar Sar L-Pro L-Pro D-Val D-Val CO CO ) H3C-tH RH (L) (L) Rt-CH3 NH NH N H2 0 CH3 CR3 Fig. 1. Actinomycin IV. Sequence of amino acids: L- Fig. 2. Circular paper chromatogram showing the actino- threonine, D-vahne (D-Val), L-prohne (L-Pro), sarcosine mycin mixtures produced by Streptomyces antibioticus. (Sar), N-methyl-L-valine (Brockmann et al. 1956; Solvent system: 10% aqueous solution of sodium o- Bullock & Johnson, 1957). Actinomycin I. 1 mole of cresotinate-di-n-butyl ether-8-tetrachloroethane (4:3: 1, proline is replaced by 1 mole of hydroxyproline (Brook- by vol.). Sequence of actinomycins is from centre to mann & Pampus, 1955). Actinomycin II. 2 moles of periphery; in all cases, the first zone just beyond the proline are replaced by 2 moles of sarcosine (Johnson & origin constitutes biologically-inactive coloured material. Mauger, 1959). Actinomycin III. 1 mole of proline is A, Actinomycin mixture produced in the glutamic acid- replaced by 1 mole of sarcosine (Johnson & Mauger, galactose medium; actinomycins I II (trace), III 1959). Actinomycin V. 1 mole of proline is replaced by (trace), IV and V; B, actinomycin mixture produced in 1 mole of 4-oxoproline (Brockmann & Manegold, 1958). the glutamic acid-galactose medium plus sarcosine Actinomycin VI. 1 mole ofD-valine is replaced by 1 mole (250,ug./ml.); actinomycins I, II, III, IV, an unidentified of D-alloisoleucine (Brockmann et al. 1956). Actinomycin component and V; C, actinomycin mixture produced in VII. 2 moles of D-valine are replaced by 2 moles of D- the glutamic acid-galactose medium plus L-valine alloisoleucine (Brockmann et al. 1956). (1000 pg./ml.); same as in A. 460 E. KATZ AND W. A. GOSS I959 Extraction of actinomycin. After a given period of incu- of the washed mycelium was then inoculated into flasks of bation, the actinomycin in 500 ml. of a fermentation glutamic acid-galactose medium and allowed to grow for a broth from a replicate series of flasks was extracted three 48 hr. period. The mycelium was harvested by centrifuging, times with butan-l-ol [20, 10, and 5% (v/v) respectively]. washed twice in 0-09 % NaCl soln. and finally suspended in The extracts were combined and the butan-l-ol was water; the final amount of mycelium was 8 mg./ml. A removed by distillation in vacuo. The crude actinomycin 20 ml. amount of this suspension was inoculated into each residue was recovered in a small volume of acetone and of a duplicate series of 250 ml. Erlenmeyer flasks contain- used directly for circular paper chromatography. If ing 20 ml. of m/15-phosphate buffer, 30 ml. of water and necessary, the material could be recovered by evaporation 10 ml. of an aqueous solution of sarcosine or L-glutamic of the acetone and then stored in the dry state in the dark acid (final concentration, 1 mM). In place of substrate, 10 ml. until used. of water was added to the control flasks. The flasks were Extraction of actinomycin from the mycelium of S. shaken at 240 rev./min. at 280. The antibiotic titre of antibioticus was accomplished in the following manner: the culture filtrates was determined daily by the disk-assay mycelium in a sample of broth was collected by suction method.
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    Interpretive Guide for Amino Acids Intervention Options LOW HIGH Essential Amino Acids Arginine (Arg) Arg Mn Histidine (His) Folate, His Isoleucine (Ile) * B6, Check for insulin insensitivity Leucine (Leu) * B6, Check for insulin insensitivity Lysine (Lys) Carnitine Vitamin C, Niacin, B6, Iron, a-KG Methionine(Met) * B6, á-KG, Mg, SAM Phenylalanine (Phe) * Iron,VitaminC,Niacin,LowPhediet Threonine(Thr) * B6, Zn Tryptophan(Trp) Trpor5-HTP Niacin, B6 Valine (Val) * B6, Check for insulin insensitivity Essential Amino Acid Derivatives Neuroendocrine Metabolism y-Aminobutyric Acid (GABA) a-KG, B6 Glycine (Gly) Gly Folate, B6,B2,B5 Serine (Ser) B6, Mn, Folate * Taurine (Tau) Tau, B6 Vit. E, Vit. C, B-Carotene, CoQ10, Lipoate Tyrosine(Tyr) Iron,Tyr,VitaminC,Niacin Cu, Iron, Vitamin C, B6 Ammonia/Energy Metabolism a-Aminoadipic Acid B6, a-KG Asparagine (Asn) Mg Aspartic Acid (Asp) a-KG, B6 Mg, Zn Citrulline (Cit) Mg, Aspartic acid Glutamic Acid (Glu) B6, a-KG Niacin, B6 Glutamine (Gln) a-KG, B6 Ornithine (Orn) Arg Mg, a-KG, B6 Sulfur Metabolism Cystine (Cys) NAC B2 Cystathionine B6 Homocystine (HCys) B6, Folate, B12, Betaine Additional Metabolites a-Amino-N-Butyric Acid a-KG, B6 B6, a-KG Alanine (Ala) * B6 Anserine Zn n-Alanine Lactobacillus and Bifidobacteria, B6 n-Aminoisobutyric Acid B6 Carnosine Zn Ethanolamine Mg Hydroxylysine (HLys) Vitamin C, Iron, a-KG Hydroxyproline (HPro) Vitamin C, Iron, a-KG 1-Methylhistidine Vitamin E, B12, Folate 3-Methylhistidine BCAAs, Vit. E, Vit. C, n-Carotene, CoQ10, Lipoate Phosphoethanolamine (PE) SAM, B12, Folate, Betaine Phosphoserine Mg Proline (Pro) a-KG Vitamin C, Niacin Sarcosine B2 * Use balanced or custom mixtures of essential amino acids Nordic Laboratiroes∙ Nygade 6, 3.sal ∙ 1164 Copenhagen K ∙ DenmarkTel: +45 33 75 1000 ∙ e-mail: [email protected] In association with ©Metametrix, Inc.
  • Amino Acid Catalyzed Direct Asymmetric Aldol Reactions: a Bioorganic Approach to Catalytic Asymmetric Carbon-Carbon Bond-Forming Reactions

    Amino Acid Catalyzed Direct Asymmetric Aldol Reactions: a Bioorganic Approach to Catalytic Asymmetric Carbon-Carbon Bond-Forming Reactions

    5260 J. Am. Chem. Soc. 2001, 123, 5260-5267 Amino Acid Catalyzed Direct Asymmetric Aldol Reactions: A Bioorganic Approach to Catalytic Asymmetric Carbon-Carbon Bond-Forming Reactions Kandasamy Sakthivel, Wolfgang Notz, Tommy Bui, and Carlos F. Barbas III* Contribution from The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 ReceiVed January 3, 2001 Abstract: Direct asymmetric catalytic aldol reactions have been successfully performed using aldehydes and unmodified ketones together with commercially available chiral cyclic secondary amines as catalysts. Structure- based catalyst screening identified L-proline and 5,5-dimethyl thiazolidinium-4-carboxylate (DMTC) as the most powerful amino acid catalysts for the reaction of both acyclic and cyclic ketones as aldol donors with aromatic and aliphatic aldehydes to afford the corresponding aldol products with high regio-, diastereo-, and enantioselectivities. Reactions employing hydroxyacetone as an aldol donor provide anti-1,2-diols as the major product with ee values up to >99%. The reactions are assumed to proceed via a metal-free Zimmerman- Traxler-type transition state and involve an enamine intermediate. The observed stereochemistry of the products is in accordance with the proposed transition state. Further supporting evidence is provided by the lack of nonlinear effects. The reactions tolerate a small amount of water (<4 vol %), do not require inert reaction conditions and preformed enolate equivalents, and can be conveniently performed at room temperature in various solvents. In addition, reaction conditions that facilitate catalyst recovery as well as immobilization are described. Finally, mechanistically related addition reactions such as ketone additions to imines (Mannich- type reactions) and to nitro-olefins and R,â-unsaturated diesters (Michael-type reactions) have also been developed.
  • Proline- and Alanine-Rich N-Terminal Extension of the Basic Bovine P-Crystallin B1 Chains

    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.