\
`
`DECEMBER 25, 1984
`
`VOlUME 259
`
`NUMBER 24
`
`ISSN 0021-9258
`JBCHA3 259(24) 14993-15689 (1984)
`
`Published by The American Society of Biological Chemists, Inc.
`
`FOUNDED BY CHRISTIAN A HERTER
`
`AND SUSTAINED IN PART BY THE CHRISTIAN A. HERTER MEMORIAL FUND
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2138 - 1/14
`
`

`
`Vol. 259, No. 24
`
`f
`The Journal
`mistry
`
`December 25, 1984
`
`Copyright © 1984 by the American Society of Biological Chemists, Inc., 428 East Preston
`
`CONTENTS*
`
`COMMUNICATIONS
`
`14993
`
`Identification and conformational changes of the
`intestinal proline carrier.
`Ernest M. Wright and Brian E. Peerce
`
`14997 Suppression of placental alkaline phosphatase bio(cid:173)
`synthesis by tunicamY,cin.
`Fumiyuki Ito and Janice Yang Chou
`
`15000 Binding of rho factor to Escherichia coli RNA po(cid:173)
`lymerase mediated by nusA protein.
`Martin C. Schmidt and Michael J. Chamberlin
`
`15003 Defect in insulin receptor phosphorylation in eryth(cid:173)
`rocytes and fibroblasts associated with severe in(cid:173)
`sulin resistance.
`Florin Grigorescu, Jeffrey S. Flier, and C. Ronald Kahn
`
`15007 6-Tridecylresorcylic acid, a novel ATPase inhibitor
`that blocks the contractile apparatus of skeletal
`muscle proteins.
`Masaki Kobayashi, Akiko Kajiwara, Masami Takahashi,
`Yasushi Ohizumi, Noboru Shoji, and Tsunematsu Take(cid:173)
`moto
`
`15010 The stereochemistry of hydrogen abstraction in vi(cid:173)
`tamin K-dependent carboxylation.
`Paulette Decottignies-Le Man~chal, Claire Ducrocq, An(cid:173)
`dn~e Marquet, and Robert Azerad
`
`15013 Characterization of verapamil binding sites in car(cid:173)
`diac membrane vesicles.
`Maria L. Garcia, Mayme J. Trumble, John P. Reuben,
`and Gregory J. Kaczorowski
`
`15017 An acetylenic mechanism-based inhibitor of dopa(cid:173)
`mine ,8-hydroxylase.
`Giovanna Colombo and Joseph J. Villafranca
`
`15021 Anglerfish preprosomatostatin II is processed to so(cid:173)
`matostatin-28 and contains hydroxylysine at resi-
`due 23.
`.
`P. C. Andrews, David Hawke, John E. Shively, and Jack
`E. Dixon
`
`15025 Comparison of the basic Escherichia coli antizyme
`1 and antizyme 2 with the ribosomal proteins S20/
`L26 and L34.
`Christos A. Panagiotidis and Evangelos S. Canellakis
`
`15028 Effects of luteinizing hormone on phosphoinositide
`metabolism in rat granulosa cells.
`JohnS. Davis, Leigh A. West, and Robert V. Farese
`
`15035 Substrate specificity of cholylglycine hydrolase for
`the hydrolysis of bile acid conjugates.
`Ashok K. Batta, Gerald Salen, and Sarah Shefer
`
`15040 Complex I binds several mitochondrial NAD-cou(cid:173)
`pled dehydrogenases.
`Balazs Sumegi and Paul A. Srere
`
`15046 Phosphate-guanosine interactions. A model for the
`involvement of guanine derivatives in autocatalytic
`reactions of ribonucleic acids.
`Gerard Lancelot and Claude Helime
`
`15051 Modification of a-bungarotoxin and cholinergic li(cid:173)
`gand-binding properties of Torpedo acetylcholine
`receptor by a monoclonal anti-acetylcholine recep(cid:173)
`tor antibody.
`Mirta Mihovilovic and David P. Richman
`
`15060 Binding of chylomicron remnants and .B-very low
`density lipoproteins to hepatic and extrahepatic li(cid:173)
`poprotein receptors. A process independent of apo(cid:173)
`lipoprotein B48.
`David Y. Hui, Thomas L. Innerarity, Ross W. Milne,
`Yves L. Marcel, and Robert W. Mahley
`
`15069 Termination sites of the in vitro DNA synthesis on
`single-stranded DNA photosensitized by proma(cid:173)
`zines.
`Marie-Paule Merville, Jacques Piette, Miguel Lopez,
`Jean Decuyper, and Albert van de Vorst
`
`15078 Na+ modulates the affinity of the lutropin chorio(cid:173)
`gonadotropin receptor.
`Karin Buettner and Mario Ascoli
`
`15085 A proton nuclear magnetic resonance study of 'Y(cid:173)
`glutamyl-amino acid cyclotransferase in human
`erythrocytes.
`Michael J. York, Philip W. Kuchel, and Bogdan E.
`Chapman
`
`15089 Proposed structure for the noncovalently associated
`heme prosthetic group of dissimilatory nitrite re(cid:173)
`ductases. Configuration of substituents of acrylo(cid:173)
`chlorin.
`Russell Timkovich, Margaret S. Cork, and Priscilla V.
`Taylor
`
`15094 Metal site cooperativity within cytochrome oxidase.
`Gay Goodman
`
`15100 Biochemical characterization of human eosinophil
`Charcot-Leyden crystal protein (lysophospholi(cid:173)
`pase).
`Peter F. Weller, DavidS. Bach, and K. Frank Austen
`
`15106
`
`Inhibition of the serine proteases leukocyte elastase,
`pancreatic elastase, cathepsin G, and chymotrypsin
`by peptide boronic acids.
`Charles A. Kettner and Ashok B. Shenvi
`
`15115 Rapid agonist-induced decrease of 1251-pindolol
`binding to ,8-adrenergic receptors. Relationship to
`desensitization of cyclic AMP accumulation in intact
`heart cells.
`Joel Linden, Amrat Patel, Arthur M. Spanier, and Wil(cid:173)
`liam B. Weglicki
`
`15123 A kinetic comparison of the processing and secre(cid:173)
`tion of the a,B dimer and the uncombined a and .B
`subunits of chorionic gonadotropin synthesized by
`human choriocarcinoma cells.
`Barry P. Peters, Raymond F. Krzesicki, Richard J. Har(cid:173)
`tle, Fulvia Perini, and Raymond W. Ruddon
`
`15131 Branch specificity of bovine colostrum and calf thy(cid:173)
`mus UDP-Gal:N-acetylglucosaminide ,B-1,4-galac(cid:173)
`tosyltransferase.
`Willem M. Blanken, Anja van Vliet, and Dirk H. van
`den Eijnden
`
`*The CONTENTS arranged by Subject Categories will be found immediately following these CONTENTS.
`Full Instructions to Authors will be found in (1984) J. Biol. Chem. 259, 1-11, and reprints may be obtained from the editorial office.
`ii
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2138 - 2/14
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`
`15136 Sequences of six genes and several open reading
`frames in the kinetoplast maxi circle DNA of Leish(cid:173)
`mania tarentolae.
`Vidal F. de la Cruz, Nicolas Neckelmann, and Larry
`Simpson
`
`15148 Inhibitor peptide of mitochondrial proton adenosine
`triphosphatase. Neutralization of its inhibitory ac(cid:173)
`tion by calmodulin.
`Peter L. Pedersen and Joanne Hullihen
`
`15154
`
`15159
`
`Interaction of diphtheria toxin with adenylyl(cid:173)
`(3',5')-uridine 3'-monophosphate. I. Equilibrium
`and kinetic measurements.
`Carleen M. Collins, Joseph T. Barbieri, and R. John
`Collier
`
`Interaction of diphtheria toxin with adenylyl(cid:173)
`(3',5')-uridine 3'-monophosphate. II. The NAD(cid:173)
`binding site and determinants Of dinucleotide affin(cid:173)
`ity.
`Carleen M. Collins and R. John Collier
`
`15163 Transient kinetic analysis of turnover-dependent
`fluorescence of 2' ,3' -0-(2,4,6-trinitrophenyl)-ATP
`bound to Ca2+-ATPase of sarcoplasmic reticulum.
`James E. Bishop, J. David Johnson, and Mervyn C.
`Berman
`
`15172 Stimulation of the purified erythrocyte Ca2+(cid:173)
`A TPase by tryptic fragments of calmodulin.
`Danilo Guerini, Joachim Krebs, and Ernesto Carafoli
`
`15178 Evidence for distinct catabolic pathways of adenine
`ribonucleotides and deoxyribonucleotides in human
`T lymphoblastoid cells.
`Jerzy Barankiewicz and Amos Cohen
`
`15182 Kinetics of synthesis, processing, and membrane
`transport of heat-labile enterotoxin, a periplasmic
`protein in Escherichia coli.
`Harmen Hofstra and Bernard Witholt
`
`15188 Purification and characterization of a lysophospho(cid:173)
`lipase from human amnionic membranes.
`Alice A. Jarvis, Carin Cain, and Edward A. Dennis
`
`15196 The nucleotide binding site detected by affinity la(cid:173)
`beling in the large T proteins of polyoma and SV 40
`viruses is distinct from their A TPase catalytic site.
`Philippe Clertant, Patrick Gaudray, Evelyne May, and
`Franr;ois Cuzin
`
`15204 Adrenergic regulation of glucose metabolism in rat
`heart. A calcium-dependent mechanism mediated by
`both a- and ,B-adrenergic receptors.
`Michael G. Clark and Glen S. Patten
`
`15212 The primary structure of Salmonella typhimurium
`HPr, a phosphocarrier protein of the phosphoenol(cid:173)
`pyruvate:glycose phosphotransferase system. A cor(cid:173)
`rection.
`Dennis A. Powers and Saul Roseman
`
`15215
`
`Inhibition of S-adenosyl-L-methionine sterol-C-24-
`methyltransferase by analogues of a carbocationic
`ion high-energy intermediate. Structure activ; ·y re(cid:173)
`lationships for C-25 heteroatoms (N, As, S) ... bsti(cid:173)
`tuted triterpenoid derivatives.
`Alain Rahier, Jean-Claude Genot, Francis Schuber,
`Pierre Benveniste, and Acharan S. Narula
`
`15224 Complete eDNA-derived amino acid sequence of
`chick muscle creatine kinase.
`Charles P. Ordahl, Gregory L. Evans, Thomas A. Cooper,
`Gisela Kunz, and Jean-Claude Perriard
`
`15228 Conformational change of sodium- and potassium(cid:173)
`dependent adenosine triphosphatase. Conforma(cid:173)
`tional evidence for the Post-Albers mechanism in
`Na+- and K+-dependent hydrolysis of ATP.
`Kazuya Taniguchi, Kuniaki Suzuki, Daitatsu Kai, Ichiro
`Matsuoka, Kinai Tomita, and Shoichi !ida
`
`iii
`
`15234 Structural elucidation of a unique macrocylic mem(cid:173)
`brane lipid from a new, extremely thermophilic,
`deep-sea hydrothermal vent archaebacterium,
`Methanococcus jannaschii.
`Paul B. Comita, Robert B. Gagosian, Henrianna Pang,
`and Catherine E. Costello
`
`15242 Multihormonal regulation of phosphoenolpyruvate
`carboxykinase gene transcription. The dominant
`role of insulin.
`Kazuyuki Sasaki, Timothy P. Cripe, Stephen R. Koch,
`Teresa L. Andreone, Daniel D. Petersen, Elmus G. Beale,
`and Daryl K. Granner
`
`15252 Ricin-like plant toxins are evolutionarily related to
`single-chain ribosome-inhibiting proteins from
`Phytolacca.
`Michael Ready, Ken Wilson, Michael Piatak, and Jon D.
`Robertus
`
`15257 Mobile domains in ribosomes revealed by proto~
`nuclear magnetic resonance.
`Cynthia A. Cowgill, Brenda G. Nichols, James W. Kenny,
`Peter Butler, E. Morton Bradbury, and Robert R. Traut
`
`15264 L(+)-Lactate binding to preparations of rat hepato(cid:173)
`cyte plasma membranes.
`Simon G. Welch, Hilary K. Metcalfe, John P. Monson,
`Robert D. Cohen, Robert M. Henderson, and Richard A.
`Iles
`
`15272 The content of long-chain free fatty acids and their
`effect on energy transduction in chromaffin granule
`ghosts.
`Eystein S. Husebye and Torgeir Flatmark
`
`15277 Estradiol-induced alteration of very-low-density li(cid:173)
`poprotein assembly. Possible competition among
`apoproteins for incorporation into nascent very(cid:173)
`low-density lipoprotein.
`Kurt W. Miller and M. Daniel Lane
`
`15287 Probing the structure of 16 S ribosomal RNA from
`Bacillus brevis.
`JoAnn Kop, Alexei M. Kopylov, Linda Magrum, Rebecca
`Siegel, Ramesh Gupta, Carl R. Woese, and Harry F. Noller
`
`15294 Effects of the specific cAMP antagonist, (Rp)-aden(cid:173)
`osine cyclic 3' ,5' -phosphorothioate, on the cAMP(cid:173)
`dependent protein kinase-induced activity of he(cid:173)
`patic glycogen phosphorylase and glycogen syn(cid:173)
`thase.
`John D. Rothermel, Nancy L. Perillo, James S. Marks,
`and Lynne H. Parker Botelho
`
`15301 Photoaffinity cross-linking of the coupling factor 1
`from Micrococcus luteus by 3' -arylazido-8-azido(cid:173)
`ATP.
`Hans-Jochen Schafer and Klaus Dose
`
`15307 Domain structure of vitronectin. Alignment of ac(cid:173)
`tive sites.
`Shintaro Suzuki, Michael D. Pi?_rschbacher, Edward G.
`Hayman, Khanh Nguyen, Yvonne Ohgren, and Erkki Ruos(cid:173)
`lahti
`
`15315 A membrane potential-sensitive Na+-H+ exchange
`system in flagella isolated from sea urchin sperma-
`tozoa.
`·
`Han Cheung Lee
`
`15320 Effects of guanyl nucleotides and rhodopsin on
`ADP-ribosylation of the inhibitory GTP-binding
`component of adenylate cyclase by pertussis toxin.
`Su-Chen Tsai, Ronald Adamik, Yasunori Kanaho, Erik
`L. Hewlett, and Joel Moss
`
`15324 Analysis of the steroid receptor of Achlya ambisex(cid:173)
`ualis.
`Robert M. Riehl and David 0. Toft
`
`15331
`
`Interaction of aspartate and aspartate-derived anti(cid:173)
`metabolites with the enzymes of the threonine bio(cid:173)
`synthetic pathway of Escherichia coli.
`Spencer L. Shames, David E. Ash, Frederick C. Wedler,
`and Joseph J. Villafranca
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`
`lV
`
`15340 Sulfmyoglobin. Resonance Raman spectroscopic
`evidence for an iron-chlorin prosthetic group.
`Laura A. Andersson, Thomas M. Loehr, Anthony R. Lim,
`and A. Grant Mauk
`
`15350 Relationship of thyrotropin-releasing hormone-in(cid:173)
`duced spike and plateau phases in cytosolic free Ca2+
`concentrations to hormone secretion. Selective
`blockade using ionomycin and nifedipine.
`Paul R. Albert and Armen H. Tashjian, Jr.
`
`15364 Phosphatidylinositol turnover in isolated soybean
`membranes stimulated by the synthetic growth hor(cid:173)
`mone 2,4-dichlorophenoxyacetic acid.
`D .. James Morre, Barbara Gripshover, Alice Monroe, and
`Jeffrey T. Morre
`
`15369 Swainsonine inhibits glycoprotein degradation by
`isolated rat liver lysosomes.
`James R. Winkler and Harold L. Segal
`
`15373 Characterization of a sulfite reductase from Desul(cid:173)
`fovibrio vulgaris. Evidence for the presence of a
`low-spin siroheme and an exchange-coupled siro(cid:173)
`heme-[ 4Fe-4S] unit.
`Boi Hanh Huynh, Ling Kang, Daniel V. DerVartanian,
`Harry D. Peck, Jr., and Jean LeGal!
`
`15377 On the mechanism of action of cytochrome P-450.
`Oxidation and reduction of the ferrous dioxygen
`complex of liver microsomal cytochrome P-450 by
`cytochrome b5.
`Denis Pompon and Minor J. Coon
`
`15386 In vivo transcription of a human antithrombin HI
`"minigene."
`Edward V. Prochownik and Stuart H. Orkin
`
`15393 Extracellular metabolism of glutathione accounts
`for its disappearance from the basolateral circula(cid:173)
`tion of the kidney.
`William A. Abbott, Richard J. Bridges, and Alton Meister
`
`15401
`
`Isolation and sequence of the structural gene for
`cytochrome c oxidase subunit VI from Saccharo(cid:173)
`myces cerevisiae.
`Richard M. Wright, Christine Ko, Michael G. Cumsky,
`and Robert 0. Payton
`
`15408 Estrogen-dependent modification of ribosomal pro(cid:173)
`teins. Effects of estrogen withdrawal on the distri(cid:173)
`bution of constitutive and hormonally regulated
`mRNAs.
`Alan W. Cochrane and Roger G. Deeley
`
`15414 Azidotyrosylcalmodulin derivatives. Specific
`probes for protein-binding domains.
`Rachel E. Kievit and Thomas C. Vanaman
`
`15425 Bacteriophage T4 gene 44 DNA polymerase acces(cid:173)
`sory protein. Sequences of gene 44 and its protein
`product.
`Eleanor K. Spicer, Nancy G. Nossal, and Kenneth R.
`Williams
`
`15433 Purification, characterization, and pituitary regu(cid:173)
`lation of the sex-specific cytochrome P-450 15/j(cid:173)
`hydroxylase from liver microsomes of untreated fe(cid:173)
`male rats.
`Catriona MacGeoch, Edward T. Morgan, James Halpert,
`and Jan-Ake Gustafsson
`
`15440 Direct evidence for the transmembrane orientation
`of the hepatic glycoprotein receptors.
`Kenneth B. Chiacchia and Kurt Drickamer
`
`15447 GTP hydrolysis by pure Nh the inhibitory regula(cid:173)
`tory component of adenylyl cyclases.
`Teresa Sunyer, Juan Codina, and Lutz Birnbaumer
`
`15452 Aspergillus contains multiple tubulin genes.
`James A. Weatherbee and N. Ronald Morris
`
`15464 Conversion of adrenergic mechanism from an a- to
`a {j-type during primary culture of rat hepatocytes.
`Accompanying decreases in the function of the in(cid:173)
`hibitory guanine nucleotide regulatory component
`of adenylate cyclase identified as the substrate of
`islet-activating protein.
`Hiroshi Itoh, Fumikazu Okajima, and Michio Ui
`
`15474 The gene encoding the {j-subunit of rat luteinizing
`hormone. Analysis of gene structure and evolution
`of nucleotide sequence.
`Larry Jameson, William W. Chin, Anthony N. Hollen(cid:173)
`berg, Albert S. Chang, and Joel F. Habener
`
`15481 Rat hepatic cytochrome P-450 isoenzyme 2c. Iden(cid:173)
`tification as a male-specific, developmentally in(cid:173)
`duced steroid 16a-hydroxylase and comparison to a
`female-specific cytochrome P-450 isoenzyme.
`David J. Waxman
`
`15491 Phenylalanyl-tRNA synthetases from sheep liver
`and yeast. Correlation between net charge and bind(cid:173)
`ing to ribosomes.
`Jean-Philippe Pailliez and Jean-Pierre Waller
`
`15497 Restriction endonuclease accessibility of the devel(cid:173)
`opmentally regulated goat ')'-, {jc-, and {jA-globin
`genes in chromatin. Differences in 5' regions which
`show unusually high sequence homology.
`Paul A. Liberator and Jerry B. Lingrel
`
`15502 Purification and properties of the activating en(cid:173)
`zyme for iron protein of nitrogenase from the pho(cid:173)
`tosynthetic bacterium Rhodospirillum rubrum.
`Leonard L. Saari, Eric W. Triplett, and Paul W. Ludden
`
`15509 Association of 1251-nerve growth factor with PC12
`pheochromocytoma cells. Evidence for internaliza(cid:173)
`tion via high-affinity receptors only and for long(cid:173)
`term regulation by nerve growth factor of both
`high- and low-affinity receptors.
`Paulette Bernd and Lloyd A. Greene
`
`15517 A novel low-activity form of carbonic anhydrase I
`in erythrocytes of patients with primary aldoste(cid:173)
`ronism. Evidence for the presence of a mixed disul(cid:173)
`fide with glutathione.
`Takahito Kondo, Naoyuki Taniguchi, Tetsuo Hirano, and
`Yoshikazu Kawakami
`
`15523
`
`Interaction of tubulin with chromatin proteins. Hl
`and core histones.
`Gilles Mithieux, Christian Alquier, Bernard Raux, and
`Bernard Rousset
`
`15532 Gapped minicircles. A novel replication intermedi(cid:173)
`ate of kinetoplast DNA.
`Peter A. Kitchin, Viiu A. Klein, Bennett I. Fein, and
`Paul T. Englund
`
`15540 The Ca2 + -pumping ATPase in skeletal muscle sar(cid:173)
`colemma. Calmodulin dependence, regulation by
`cAMP-dependent phosphorylation, and purifica(cid:173)
`tion.
`Marek Michalak, Konrad Famulski, and Ernesto Carafoli
`
`15548 Analysis of gene expression during differentiation
`of adipogenic cells in culture and hormonal control
`of the developmental program.
`Alger B. Chapman, David M. Knight, Barbara S. Dieck(cid:173)
`mann, and Gordon M. Ringold
`
`15556 Human proapolipoprotein A-II is cleaved following
`secretion from Hep G2 cells by a thiol protease.
`Jeffrey I. Gordon, Harold F. Sims, Celina Edelstein,
`Angelo M. Scanu, and Arnold W. Strauss
`
`15564 Comparison of myosin heavy chains in atria and
`ventricles from hyperthyroid, hypothyroid, and eu(cid:173)
`thyroid rabbits.
`Richard A. Chizzonite, Alan W. Everett, Gwen Prior, and
`Radovan Zak
`
`15460 In vitro acylation of the transferrin receptor.
`Mohammed Adam, Angel Rodriguez, Claire Turbide,
`James Larrick, Edward Meighen, and Rose M. Johnstone
`
`15572 A general procedure for the end labeling of proteins
`and positioning of amino acids in the sequence.
`Daniel G. Jay
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`
`15579 Morphology of ~roteolip<?somes reconsti.tut~d w~th
`purified lac earner protem from Escherzchza colz.
`M. Joseph Costello, Paul Viitanen, Nancy Carrasco,
`David L. Foster, and H. Ronald Kaback
`15587 Phosphorylation of purified cardiac muscle C-pro(cid:173)
`tein by purified cAMP-dependent and endogenous
`Ca2+ -calmodulin-dependent protein kinases.
`H. Criss Hartzell and David B. Glass
`15597 Translationally associated helix-destabilizing ac(cid:173)
`tivity in rabbit reticulocyte lysate.
`Stephen A. Liebhaber, Faith E. Cash, and Susan H.
`Shakm
`Induction by cAMP of the mRNA encoding th~ cy(cid:173)
`tosolic form of phosphoenolpyruvate carboxykmase
`(GTP) from the chicken. Identification and charac(cid:173)
`terization of a cDN A clone for the enzyme.
`Yaacov Had, Sidney M. Morris, and Richard W. Hanson
`
`15603
`
`v
`
`15609 The gene encoding the cytosolic form of phosphoen(cid:173)
`olpyruvate carboxykinase (GTP) from the chicken.
`Yaacov Hod, Heeja Yoo- Warren, and Richard W. Han(cid:173)
`son
`
`ADDITIONS AND CORRECTIONS
`
`15615 Differential compartmentation of magnesium and
`calcium in murine S49 lymphoma cells. Vol. 259
`(1984)12184-12192.
`Robert D. Grubbs, Stephen D. Collins, and Michael E.
`Maguire
`
`15616 Cumulative Author Index. Numbers 1 through 24.
`
`15633 Subject Index.
`
`GENERAL INFORMATION FOR 1985
`
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`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`'© 1984 by The American Society of Biological Chemists, Inc.
`
`Vol. 259, No. 24, Issue of December 25, pp. 15106-15114, 1984
`Printed in U.S.A.
`
`Inhibition of the s·erine Proteases Leukocyte Elastase, Pancreatic
`Elastase, Cathepsin G, and Chymotrypsin by Peptide Boronic Acids*
`
`Charles A. Kettner and Ashok B. Shenvi
`From the Central Research and Development Department, E. I. DuPont de Nemours and Company, Experimental Station,
`Wilmington, Delaware 19898
`
`(Received for publication, May 21, 1984)
`
`Three a-aminoboronic acid-containing analogs of
`good peptide substrates for serine proteases were syn(cid:173)
`thesized, MeO-Suc-Ala-Ala-:Pro-boro-Phe-OH, MeO(cid:173)
`Suc- Ala- Ala- Pro- boro- Ala- OH, andMeO- Sue- Ala(cid:173)
`Ala-Pro-boro-Val-OH. They were effective inhibitors
`of chymotrypsin,_ cathepsin G, and both leukocyte and
`pancreatic elastase at nanomolar concentrations
`(0.10-20 nM). Except for cathepsin G, inhibition was
`not simply competitive, but showed kinetic properties
`corresponding to the mechanism for slow-binding in(cid:173)
`hibition, i.e. E + I ~ EI ~ EI*, where EI and EI* are
`enzyme-inhibitor complexes and EI* is more stable
`than EI. This type of inhibition has not been observed
`previously for synthetic inhibitors or serine proteases
`and in this study it was observed only for peptide
`boronic acids which satisfy the primary specificity
`requirements of the protease.
`
`Leukocyte elastase and cathepsin G are serine proteases
`associated with the granular fraction of polymorphonuclear
`leukocytes which hydrolyze multiple bonds of protein sub(cid:173)
`strates. Particular emphasis has been placed on the study of
`leukocyte elastase due to its strong implication in diseases
`such as emphysema and septicemia. Its role in emphysema is
`in the aberrant proteolysis of lung elastin (Mittman (1972;
`Turino et al., 1974; Hance and Crystal, 1975) while in septi(cid:173)
`cemia, elevated plasma levels of elastase associated with in(cid:173)
`fection suggest that it is involved in nonspecific degradation
`of plasma proteins (Jochum et al., 1983). The role of cathepsin
`G in disease processes is less clear, but its ability to degrade
`proteoglycans and elastin suggest that it may also be involved
`in emphysema (Reilly and Travis, 1980). Cathepsin G also
`liberates the hypertensive peptide angiotensin II from angi(cid:173)
`otensin I (Reilly et al., 1982) and from angiotensinogen (Win(cid:173)
`troub et al., 1984); however, the physiological significance of
`these reactions is unclear. Therefore, specific inhibitors of
`leukocyte elastase and cathepsin G may be useful therapeu(cid:173)
`tically in the control of emphysema and septicemia as well as
`in determining the physiological significance of other reac(cid:173)
`tions catalyzed by cathepsin G and elastase.
`A number of synthetic inhibitors of leukocyte and pan(cid:173)
`creatic elastase and of cathepsin G and chymotrypsin are
`known. These inhibitors include peptide chloromethyl ke(cid:173)
`tones (Powers et al., 1977), azapeptides (Powers and Carroll,
`1975), trifluoroacetyl peptides (Renaud et al., 1983), arylsul(cid:173)
`fonyl fluorides (Yoshimura et al., 1982), and heterocyclic
`
`* The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked "advertisement" in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`
`agents (Zimmer:rn.an et al., 1980; Teshima et al., 1982). Ki
`values for the more effective inhibitors obtained in these
`studies range from 0.10 to 0.010 f.LM (see Teshima et al., 1982,
`for comparisons).
`The approach we have taken in a search for more selective
`and effective inhibitors of the leukocyte proteases pancreatic
`elastase and chymotrypsin has been to prepare peptide bo(cid:173)
`ronic acids. This group of compounds takes advantage of
`binding in the P1-P5 sites (using the nomenclature of Schech(cid:173)
`ter and Berger, 1967) and of the binding of the boronic acid
`which can potentially act as a "transition state analog." Se(cid:173)
`lection of the residues in the PcP5 binding sites of the peptide
`boronic acids was based on the amino acid sequence of the
`better substrates of these enzymes from the extensive speci(cid:173)
`ficity studies of Zimmerman and Ashe (1977), Nakajima et
`al., (1979), and McRae et al. (1980). The proposed mechanism
`of binding of peptide boronic acids is shown in Fig. 1.
`The serine proteases chymotrypsin and subtilisin have been
`shown to be inhibited by aromatic boronic acids (Koehler and
`Lienhard, 1971; Lindquist and Terry, 1974). The crystal struc(cid:173)
`ture of subtilisin complexed to either phenylethane boronic
`acid or phenyl boronic acid has been determined (Matthews
`et al., 1975). In the crystal structure, the trigonal boronic acid
`exists as a transition state-like, tetrahedral adduct with the
`active site serine of the protease. One -OH of boron occupies
`the oxyanion hole and the other -OH corresponds to the
`expected position of the departing NH- group for normal
`hydrolysis.
`Matteson et al. (1981) have further demonstrated the po(cid:173)
`tential of boronic acids as serine protease inhibitors by the
`preparation of the boronic acid analog of N-acetyl-phenylal-
`
`Substrate Hydrolysis
`
`!Jo
`peptide- NH-CH-C'I
`I
`\
`R
`NH,......
`OH
`I s~rl .
`
`Boronic Acid BindinQ
`
`oe
`I
`peptide-NH-CH-C-NH-
`1
`I
`R
`0
`
`I S~r I
`
`OH
`I
`peptide -NH-CH-B
`~
`'oH
`OH
`I S~rl
`FIG. 1. Scheme for the mechanism of binding for peptide
`boronic acids.
`
`OH
`le
`peptide- NH-CH- B-OH
`I
`I
`R
`0
`1 ser 1
`
`15106
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2138 - 6/14
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`
`Inhibition of Serine Proteases
`
`15107
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`anine, (R)-acetamido-2-phenylethane boronic acid. 1 They
`have shown that this compound is effective in the micromolar
`concentration range and is several orders of magnitude more
`effective than the aromatic boronic acids in the inhibition of
`chymotrypsin. In addition, the synthetic route developed by
`these workers was instrumental for us in the synthesis of
`other a-amino boronic acids used in the preparation of peptide
`boronic acids for the present study.
`
`EXPERIMENTAL PROCEDURES
`
`Bovine a-chymotrypsin, 49 units mg-I, was obtained from Wor(cid:173)
`thington. Stock solutions were prepared in 1.0 mM HCl and were
`stored for no longer than three days at 4 °C. Cathepsin G, was a
`generous gift from Dr. James Travis, University of Georgia, and was
`stored frozen in 0.10 M pyridine acetate buffer, pH 5.5, and 0.40 M
`NaCl. Porcine pancreatic elastase am~ human sputum leukocyte
`elastase were obtained from Elastin Products, Pacific, MO. Human
`leukocyte elastase was also prepared by the procedure of Baugh and
`Travis (1976). Solutions of lyophilized pancreatic elastase were pre(cid:173)
`pared in 0.05 M sodium ·acetate buffer, pH 5.5, containing 0.40 M
`NaCI, and were then diluted to 50% glycerol (w/w) and stored at
`-20 a c. Stock solutions of leukocyte elastase (1.0 mg ml-1
`) were
`prepared in 0.05 M sodium acetate buffer, pH 5.5, containing 0.40 M
`NaCl and stored at -20 °C.
`Chymotrypsin and cathepsin G were assayed with the substrate
`Suc-Ala-Ala-Pro-Phe-p-nitroanilide reported by Nakajima et al.
`(1979). More sensitive assays of chymotrypsin used Suc-Ala-Ala-Pro(cid:173)
`Phe-7-amidino-4-methylcoumarin (Enzyme Systems Products). Pan(cid:173)
`creatic elastase was assayed with Suc-Ala-Ala-Ala-p-nitroanilide
`(Sigma) as a substrate (Bieth et al., 1974). Leukocyte elastase was
`assayed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (Nakajima et
`al., 1979). Assay solutions contained 1% dimethyl sulfoxide for chy(cid:173)
`motrypsin and leukocyte elastase and 5% dimethyl sulfoxide for
`cathepsin G.
`The hydrolysis of p-nitroanilide substrates was monitored at 405
`nm on a Cary 219 spectrophotometer. Hydrolysis of 7-amidino-4-
`methylcoumarin substrates was monitored by excitation at 380 nm
`and measuring emission at 460 nm on a Perkin-Elmer 650-40 fluo(cid:173)
`rometer.
`The concentrations of pancreatic elastase, leukocyte elastase, and
`chymotrypsin were estimated by the titrimetric procedure described
`by Morrison (1969) using the most effective peptide boronic acid
`inhibitor. See data in Fig. 6 for an example. The concentration of
`cathepsin G was based on weight.
`All kinetic constants were determined in 0.10 M sodium phosphate
`buffer, pH 7.5, containing 0.50 M NaCl at 25 ac. K; values for peptide
`boronic acids were determined from double reciprocal plots of velocity
`versus substrate concentration by the method of Lin weaver and Burk.
`Data were fitted to the best straight lines by the least-squares method.
`In all cases the inhibitor concentration was at least 5-fold greater
`than the enzyme concentration. Five or six concentrations of sub(cid:173)
`strate were chosen so that values of reciprocal concentrations were
`evenly proportioned. The substrate concentration ranges and kinetic
`constants for substrate hydrolysis are given in Appendix I (Table I).2
`For reactions in which progressive, time-dependent inhibition was
`
`1 (R)-Acetamido-2-phenylethane boronic acid is an analog of N(cid:173)
`acetyl-phenylalanine in which the -COOH group is replaced by
`-B(OHh We have abbreviated this compound and other a-amino
`boronic acids by the prefix "boro" and the name of the corresponding
`amino acid. All amino acids are in the L configuration unless specified.
`The designation of L for the· configuration of a-amino boronic acids
`is that of naturally occurring L-amino acids. Other abbreviations used
`are: Sue, succinyl, MeO-Suc, methoxysuccinyl; Hepes, N-2-hydroxy(cid:173)
`ethylpiperazine-N1-2-ethanesulfonic acid; MS, mass spectrum; Me,
`methyl; Bz, benzyl; i-Pr, iso-propyl; Boc, t-butyloxycarbonyl.
`2 Portions of this paper (including Appendice