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`

`BICHAW 25(15) 4175—4472 (1986)
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`
`
`ISSN 0006-2960
`
`
`Registered in US. Patent and Trademark (We:
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`Copyright “'86 by the American Chemical Society
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`a biweekly publication of the American Chemical Society
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`July 29, 1986
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`
`
`ACCELERATED PUBLICATIONS
`
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`
`Purification of a Protein C Activator from the Venom of the Southern Copperhead Snake (Agkistrodon
`
`
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`
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`contortrix contortrix)
`
`
`Janet D. Klein and Frederick J. Walker“
`
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`
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`
`
`ARTICLES
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`N—(6-Phenylhexyl)-S-chloro-l-naphthalenesulfonamide, a Novel Activator of Protein Kinase C
`
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`Masaaki Ito, Toshio Tanaka. Masaki Inagaki. Koji Nakant‘shi. and Hiroyoshi Hidalca‘
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`Medium-Chain Aeyl Coenzyme A Dehydrogenase from Pig Kidney Has Intrinsic Enoyl Coenzyme A
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`I-Iydratase Activity
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`Sze—Mei Lon, Pat Powell, Hermann Bnettner, Sandro Ghisla, and Colin Thorpe'
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`Noncompetitive and Irreversible Inhibition of Xanthine Oxidase by Benzimidazole Analogues Acting at the
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`Functional Flavin Adenine Dinucleotide Cofactor
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`Edward B. Skibo
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`Crystal Structure of a Novel Trimethoprim-Resistant Dihydrofolate Reductase Specified in Escherichia coli
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`by R-Plasmid R67
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`David A. Motthews.‘ S. L. Smith. D. P. Baccanari. J. J. Burchall. S. J. Outlay, and J. Kraut
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`Characterization of Phenylalanine Hydroxylase
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`L. M. Bloom, S. J. Benkooic,‘ and Betty Jean Gaffney
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`A Spectral Study of Cobalt(II)-Substituted Bacillus cereus Phospholipase C
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`Roy Bicknell,‘ Graeme R. Hanson, Barton Holmquist, and Clive Little
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`Novel Preparation of Functional Sindbis ViIOSOmes
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`Ronald K. Schem‘e
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`fi-Elimination of Indole from L-Tryptophan Catalyzed by Bacterial Tryptophan Synthase: A Comparison between
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`Reactions Catalyzed by Tryptophanase and Tryptophan Synthase
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`Syed Ashrafuddin Ahmed, Brian Martin, and Edith Wilson Miler‘
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`Isomerization of (SS)-2.3-Dihydro-5-fluoro-L-tryptophan and of S—Fluoro-L-tryptophan Catalyzed by Tryptophan
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`Synthase: Studies Using FluOrine-l9 Nuclear Magnetic Resonance and Difference Spectroscopy
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`Edith Wilson Miler} Robert S. Phillips. Herman J. C. Yell. and Louis A. Cohen
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`Role of Head Group Structure in the Phase Behavior of Amino Phospholipids.
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`Lamellar Phases of Saturated Phosphatidylethanolamine Analogues
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`John R. Siluius.‘ Pamela M. Brown. and Timothy J. O’Leary
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`I. Hydrated and Dehydrated
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`Pamela M. Brown, John Steers, Sek Wen Hui, Philip L. Yeagle, and John R. Siloius"
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`Chemical Modification of Tyrosine Residues in p-Hydmxybenzoate Hydroxylase from Pseudomonas fluorescens:
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`Assignment in Sequence and Catalytic Involvement
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`Robert A. Wijnands, Wicker J. Weijer. Franz Mullen" Peter A. Jekel. Willem J. H. van Berkel,
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`and Jeep J. Beintema
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`Role of Head Group Structure in the Phase Behavior of Amino Phospholipids. 2. Lamellar and Nonlamellar
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`Phases of Unsaturated Phosphatidylethanolamine Analogues
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`Page 3 of 15
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`4A BIOCH-aM-ISTRY, VOL. 25, NO.
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`:5,
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`I986
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`4268
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`Structural Heterogeneity of the a Subunits of the Nicotinic Acetylcholine Receptor in Relation to Agonist
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`Affinity Alkylation and Antagonist Binding
`Manohar Ratnam. William Gullick, Joachim Spiess, Kee Wan, Manuel Criaalo, and
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`Jon Lindstrom“
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`Structural Characterization of the ATP-Hydrolyzing Portion of the Coated Vesicle Proton Pump
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`Michael Forgoc“ and Michael Bertie
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`Estimated Conformation, Orientation, and Accumulation of Dynorphin A-(l-l3)-tridecapeplide on the Surface of
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`Neutral Lipid Membranes
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`Robert Schwyzer
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`Use of Cytochrome P4150m To Measure CholesteroluLipid Interactions
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`Victoria L. Stevens, J. David tambeth, and Alfred H. Merrill, Jr.‘
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`Human CI Inhibitor: Primary Structure, cDNA Cloning, and Chromosomal Localization
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`Susan Clark .Bock.‘K Karen Skrioer. Egon Nielsen, Hans-Christian Thogersert, Bjorn Wiman,
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`Virginia H. Donaldson. Roger L. Eddy. Jean Marriaari, Elzbieta Radziejewska. Robert Huber.
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`Thomas 8. Shows. and Stafian Magnassoa“
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`Iron-Containing Metallocenes as Active Site Directed Inhibitors of the Proteinase That Cleaves the
`NHz-Terminal Propeptides from Type I Procoilagen
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`Kenneth E. Dombrowslci. John E. Shears, and Darwin J. Prockop"
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`Amino Acid Sequence of a Basic Agkistrodon holy: blomhofiii Phospholipase A2. Possible Role of
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`NHz-Terminal Lysines in Action on Phospholipids of Escherichia coli
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`Steven Forst, Jerrold Weiss. Peter Blackburn, Bias FraagiOne. Fernando Gom‘. and Peter Elsbach‘
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`Subunit Structure of the Fatty Acid Reductase Complex from Photobacteriam phosphoream
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`lee Wall and Edward A. Meighen"
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`Purification of a Benzo[a]pyrene Binding Protein by Affinity Chromatography and Photoaffinity Labeling
`Sheila Collins and Michael A. Marlena"
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`Redox Properties and Mossbauer Spectroscopy of Azotobacter oinelandii Bacterioferritin
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`G. D. Watt,‘ R. B. Frankel, G. C. Papaeflhymiou, K. Spartaliaa, and E. I. Stiefel
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`Reassignment of the Guanine-Binding Mode of Reduced Mitomycin C
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`Maria Tomasz.' Roselyn Lipman, Gregory L. Verdiae. and Kojt' Nakanishi“
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`Production of Dihydrothymidine Stereoisomers in DNA by y-Irradiation
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`Eileen A. Furlong, Timothy J. Jorgensert, and William D. Henner"
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`Nucleotide Sequence Binding Preferences of Nogalamycin Investigated by DNase I Footprinting
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`Keith R. Fox‘ and Michael J. Waring
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`Isolation of a Multifunctional Protein with Aminoimidazole Ribonucleotide Synthetase. Glycinamide
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`Ribonucleotide Synthetase. and Glycinamide Ribonucleotide Transformylase Activities: Characterization of
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`Aminoimidazole Ribonucleotide Synthetase
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`J. L. Schrimsher, F. J. Schendel. and J. Stubbe“
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`Purification and Characterization of Aminoimidazole Ribonucleotide Synthetase from Escherichia coli
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`J. L. Schrimsher, F. J. Schendel, J. Stubbe,‘ and J. M. Smith
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`Covalent Modification of the Inhibitor Binding Site(s) of Escherichia coli ADP-Glucose Synthetase: Specific
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`Incorporation of the Photoaffinity Analogue B-Azidoadenosine S’-Monophosphate
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`Charles E. Larsen and Jack Preirs’
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`Partial Reversal of a2u Globulin Gene Expression by Thyroxine in the Liver of Diabetic Rats
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`C. V. Ramona Murry, William F. Demyaa. Baadana Chatterjee. and Ann: K. Roy“
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`Alteration of Intramolecular Disulfides in Insulin Receptor/Kinase by Insulin and Dithiothreitol:
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`Potentiates the Apparent DithiothreitoI-Dependent Subunit Reduction of Insulin Receptor
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`Peter A. Wilden, Timothy R. Boyle, Michael L. Swanson, laurel J. Sweet, and Jeffrey E. Pessia'
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`Insulin
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`Biochemical Signal Transmitted by Fc Receptor for Immunoglobulin G2,, of a Murine Macrophage-like Cell Line.
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`P388Dl: Mode of Activation of Adenylate Cyclase Mediated by Immunoglobulin Gh Binding Proteins
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`Rafael Fernandez-Botran and Truneo Suzuki‘
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`Raphael Zia‘overzki. Marry Bartholdi, Donna Arndt- Jooin, and Thomas M. Josie“
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`Rotational Dynamics of the Fc Receptor for Immunoglobulin E on Histamine-Releasing Rat
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`Basophilic Leukemia Cells
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`Page 4 of 15
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`

`

`BIOCHEMISTRY, VOL. 25, N0. 15, 1986
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`SA
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`.I
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`Ca2+ Binding to Chromaffin Vesicle Matrix Proteins: Effect of pH, Mg“, and Ionic Strength
`Felicitas U. Retffen and Manfred Gratzl‘
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`Apolipoprotein C-III/Sphingomyelin Recombinants: Formation, Isolation, and Characterization
`Tareq Y. Ahmad. John R. Guyton. James T. Sparrow. and Joel D. Morrisett“
`
`X
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`Human Lysosomal Sphingomyelinase: Substrate Efficacy of Apolipoprotein/Sphingomyelin Complexes
`Tareq Y. Ahmad, Arthur L. Beaudet. James T. Sparrow, and Joel D. Morrisett’
`
`Alternative Carbon Monoxide Binding Modes for Horseradish Peroxidase Studied by Resonance
`Raman Spectroscopy
`Ruby Euangelista-Kirkup, Giulietta Smulevich, and Thomas G. Spiro‘l
`
`Raman and Infrared Spectra of Cytochrome c Peroxidase-Carbon Monoxide Adducts in Alternative
`Conformational States
`
`Giulietta Smulevich, Ruby Evangelista-Kirkup, Ann English, and Thomas G. Spiro‘
`
`Identification of Amino Acid Residues Photolabeled with 2-Azido[a-”P]adenosine Diphosphate in the [3 Subunit
`of Beef Heart Mitochondrial Fl-ATPase
`Jérome Garin,‘ Franqois Boulay, Jean Paul Issartel, Joel Lunardi. and Pierre V. Vignais
`
`Menadione- (2-Methyl-l,4-naphthoquinone-) Dependent Enzymatic Redox Cycling and Calcium Release
`by Mitochondria
`Balz Frei, Kaspar H. Winterhalter, and Christoph Richter’
`
`Chromatographic Analysis of the Chiral and Covalent Instability of S-Adenosyl-L-methionine
`Jerald L. Hoffman
`
`Study of the Interaction of Escherichia coli Methionyl-tRNA Synthetase with tRNA’Met Using Chemical and
`Enzymatic Probes
`Heike Pelka and LaDonne H. Schulman‘
`
`Binding of N-Acetylgalactosamine-Specific Lectins to Spin-Labeled Galactosamine Derivatives
`Lawrence J. Berliner.‘ Giovanni Musci. Mary Maliarik. Nike R. Plea-sax. and Irwin J. Goldstein
`
`Kinetic Comparison of Ricin Immunotoxins: Biricin Conjugate Has Potentiated Cytotoxicity
`Jon W. Marsh‘ and David M. Neville. Jr.
`
`Telephone toll free 800-424-6747.
`
`Surface Properties of l.2-Dipalmitoyl-3-acyl-sn-glycerols
`David A. Fahey and Donald M. Small‘
`
`There is no supplementary material for this issue.
`
`‘ In papers with more than one author, the asterisk indicates the name of the author to
`whom inquiries about the paper should be addressed.
`
`This publication is available in
`four formats:
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`online 0 microfilm o microfiche 0 print
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`For more information, contact your information specialist, or
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`American Chemical Society, Sales Office, 1155 Sixteenth Street, NW, Washington, DC 20036.
`
`Page 5 of 15
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`

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`4292
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`Biochemistry 1986, 25, 4292-4301
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`Tschesche, R., & Wolff, G. (1963) Tetrahedron 19, 621—934.
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`Vandenbeuvel, F. A. (1963) J. Am. 0i! Chem. Soc. 40,
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`455—471.
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`van Dijek, P. W. M. (1979) Biochim. Biophys. Add 555,
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`89—101.
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`van Dijck, P. W. M., DeKruijff, B., van Deenen, L. L. M.,
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`De Gier, J ., & Demo], R. A. (1976) Biochim. Biophys. Acid
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`455, 576—587.
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`Wattenberg, B. W., & Silbert, D. F. (1983) J. Biol. Chem.
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`258, 2284-2289.
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`Yeaglc, P. L.. Hutton. W. C., Huang, C., & Martin, R. B.
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`(1975) Proc. Natl. Acad. Sci. U.S.A. 72, 34774481.
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`Yedgar, S., Barenholz, Y., & Cooper, V. G. (1974) Dion-him.
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`Biophys. Acio 363, 98—111.
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`Human Ci Inhibitor: Primary Structure, cDNA Cloning, and Chromosomal
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`Localizationl
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`Susan Clark Bock,” Karen Skriver,i Egon Nielsen,g Hans-Christian Thogersen,” Bjorn Wiman,‘
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`Virginia H. Donaldson} Roger L. Eddy,V Jean Marrinan,1 Elzbieta Radziejewska.t Robert Huber,o
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`Thomas B. Shows,v and Staffan Magnusson "i
`The Rockefeller University. New York, New York 10021. Department of Molecular Biology. University of Aarhus. Bit-8000.
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`Aarhus C, Denmark. Department of Clinical Chemistry, Karolinska Hospital, S-l040l Stockholm, Sweden, Children's Hospital
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`Research Foundation, Cincinnati, Ohio 45229, Roswell Park Memorial Institute, Buflol’o. New York 14263, and
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`Max—Plonck-lnsritur fuer Biochemie, 8033 Martinsried. FRG
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`Received April H, 1986: Revised Manuscript Received May 8. 1986
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`0006-2960 86 0425-4292$01.50 0 © 1986 American Chemical Societ
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`ABSTRACT: The primary structure of human C1 inhibitor was determined by peptide and DNA sequencing.
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`The single-chain polypeptide moiety of the intact inhibitor is 478 residues (52 869 Da), accounting for only
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`51% of the apparent molecular mass of the circulating protein (104 000 Da). The positions of six gluco-
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`samine-based and five galactosamine-based oligosaccharides were determined. Another nine threonine rmidues
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`are probably also glycosylated. Most of the carbohydrate prosthetic groups (probably 1'!) are located at
`the amino-terminal end (residues l—120) of the protein and are particularly concentrated in a region where
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`the tetrapeptide sequence Glx-Pro-Thr-Thr, and variants thereof, is repeated 7 times. No phosphate was
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`detected in C1 inhibitor. Two disulfide bridges connect cysteine-101 to cysteine—406 and cysteine-108 to
`cysteine-183. Comparison of the amino acid and cDNA sequences indicates that secretion is mediated by
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`a 22—residue signal peptide and that further proteolytic processing does not occur. Cl inhibitor is a member
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`of the large serine protease inhibitor (serpin) gene family. The homology concerns residues 120 through
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`the C-tertninus. The sequence was compared with those of nine other scrpins, and conserved and nonoonserved
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`regions correlated with elements in the tertiary structure of al-antitrypsin. The C1 inhibitor gene maps
`to chromosome 1 1, pl 1.2-q13. C1 inhibitor genes of patients from four hereditary angioneurotic edema
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`kindreds do not have obvious deletions or rearrangements in the Cl inhibitor locus. A HgiAI DNA
`polymorphism, identified following the observation of sequence variants, will be useful as a linkage marker
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`in studies of mutant Cl inhibitor genes.
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`Ci inhibitor is a highly glycosylated 104 000-13211 plasma
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`protease inhibitor that can inhibit components of the com-
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`plement, coagulation, fibrinolytic. and kinin-releasing systems.
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`It was first purified in 1961 (Pensky ct 9.1., 1961) and later
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`(Penalty 8r. Schwick, 1969) found to be immunologically
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`identical with a previously characterized aZ-neuramino-
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`glycoprotein (Schultze et al., 1962). CT inhibitor has been
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`shown to inhibit macromolecular C1, the Cls and Clr sub-
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`fThis work was supported by USPHS Grants HL-l5690 (V.H.D.),
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`HL-lfilSS (SAIL), HL-307I2 (S.C.B.), and FIB-05196 and Gilli-20454
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`(T.B.S.) and funds from the Danish Science and Medical Research
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`Councils (S.M.), the Danish Cancer Society (SM. and KS), and the.
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`American Heart Association (83-1202, S.C.B.).
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`‘ Correspondence should be addressed to these authors.
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`3The Rookel'eller University.
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`IUniversity of Aarhus.
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`lPresent address: MRC Laboratory of Molecular Biology. Cam-
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`bridge, England CBZ 20H.
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`‘- Karolinska Hospital.
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`*Children‘s Hospital Research Foundation.
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`“Roswell Park Memorial Institute.
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`"Max-Planck-Institul flier Biochemie.
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`components of the first component of complement (Ratnoff
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`& Lepow, 1957; Levy & Lepow, 1959; Lepow & Leon, 1962:
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`Gigli et 3]., 1968; Pensky et al., 1961; Ratnoff et al., 1969).
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`factors X1121 and X13 (Forbes et a1., 1970), plasma kaliikrein
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`(Ramoff et al., 1969: Giin et al., 1970), and plasmin (Ratnoff
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`ct 8.1., 1969). Like other serine protease inhibitors [serpins
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`(Carroll, 1984)] of the antithrombin III-al-antitrypsin family
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`(Petersen et 31., 1979), C1 inhibitor reacts with target proteases
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`to form proteolytically inactive, stoichiometric 1:1 complexw
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`that are stable during NaDodSO4—PAGE (Harpel & Cooper.
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`1975; Sim et al., 1979; Sim et al., 1980) under reducing
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`conditions (Nilsson et al., 1983). To improve understanding
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`of how Ci inhibitor regulates diverse plasma scrine proteases.
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`we have determined its sequence and covalent structure. To
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`address questions concerning the evolution and structure of
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`‘ Abbreviations: bp. base pairs; kb, kilobase; Da, dalton; serpin. serine
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`protease inhibitor; NaDodSO.. Sodium dodecyl sulfate: PAGE. I301!"
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`acrylamide gel electrophoresis; HANE, hereditary angioneurotic edema:
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`A, angstrom; DNS, S-(dimethylamino)naphthalenesulfonyl; RFLP. rc-
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`slriction fragment length polymorphism; HPLC, high-performance lin-Ild
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`chromatography: PTH, phenylthiohydantoin.
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`Page 6 of 15
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`

`

`" imam c-i INHIBITOR
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`VOL. 25, no. 1-5, was
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`'4293
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`-_
`, we compared the Cl inhibitor sequence with those of
`nine other family members.
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`Cl inhibitor deficiency is inherited as an autosomal dom-
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`inant trait in hereditary angioneurotic edema (HANE) which
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`is associated with bouts of localized, increased vascular
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`permeability (Donaldson & Evans, 1963). Although all
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`persons with HANE are deficient in C1 inhibitor function,
`the molecular defects are heterogeneous (Rosen et al., 1971;
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`Donaldson et al., 1985).
`in order to understand the genetic
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`lesions responsible for hereditary angionwrotic edema, we have
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`isolated a full length Cl inhibitor cDNA, mapped the Cl
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`inhibitor locus to human chromosome 11, p11.2-ql3. and
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`initiated studies on normal and abnormal C1 inhibitor genes.
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`MATERIALS AND METHODS
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`Protein Purification. Cl inhibitor was isolated from human
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`plasma by precipitatiou with poly(ethylene glycol), chroma-
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`tography on DEAE-cellulose and hexyl-Sepharose (Nilsson
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`& Wiman, 1982). and gel filtration in 0.1 M NH4HC03, pH
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`8.3, on Sepharose 6B. The resulting single-chain material was
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`fully active against Cls (Chapuis et al., 1977) and migrated
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`with an apparent molecular mass of 105000 Da in NaDod-
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`SOrPAGE. Phosphate was determined by a modified
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`Fiske—Subbarow method (Arnes, 1966). The activation pep-
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`tide was separated from the Cls—Cl inhibitor complex by gel
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`filtration on Sephacryl 5—300 in 40 mM sodium phosphate,
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`0.1 M NaCl, 0.1 M NaNJ, and 0.1% NaDodSO4, pH 7.3.
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`Amino Acid Sequencing. Cl inhibitor was degraded
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`chemically or enzymaticaily as indicated in Figure 1a. For
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`digestion with pepsin, the protein was dissolved in 99% formic
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`acid and then diluted to 5%; the enzyme]substrate ratio was
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`1/ 100 w/w, and the digest was incubated at room temperature
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`for 3 h. Carbohydrate-rich peptides were treated with tri-
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`fluoromethanesulfonic acid (Edge et al., 1981) or alkaline
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`sodium borohydride (Spiro & Bhoyroe, 1974). Peptides were
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`purified by an initial gel filtration on Sephadex G-SDF or
`Sephacryl 5-200, mostly in 0.1 M NH4HC03, followed by
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`ion-exchange chromatography on DEAE-Sephacel using a.
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`linear gradient of 001—1 .0 M NH4HC03. Final purification
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`was achieved by reversed-phase HPLC on a Hewlett-Packard
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`1084B liquid chromatograph. Peptides were sequenced on an
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`Applied Biosystems Model 470A (using the chemicals and the
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`02:1 vac program supplied by the manufacturer) or on a
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`Bookman 890C sequenator. Amino acid analysis was per-
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`formed on a Beckman 121MB instrument.
`In addition to the
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`systems commonly used in our laboratory (Skorstengaard et
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`81., 1982, 1984), columns of Vydac C4 (with elution gradients
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`of 2-propanol and triethylamine, pH 5.2, in 0.1% CF3COOH)
`were also employed for HPLC of peptides and glycopeptides.
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`Carbohydrate Determination. N-Glyoosylated Asn residues
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`were assigned when the PTH derivative was obtained in much
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`less than normal yield and the presence of aspartic acid and
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`a ninhydrin-positive glucosamine peak (eluting near isoleucine
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`and leucine) was observed in the amino acid chromatogram.
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`In addition, Asn residues 59, 216, and 231 were also identified
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`as DNS-Asp in “full” yield when the DNS—Edrnan sequencing
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`method was used (Gray, 1967). O-Glycosylated threonine or
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`serine residues were assigned on the corresponding combined
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`criteria of low yield (or no yield) of PTH derivatives and the
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`presence of galactosamine and threonine or serine in the hy-
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`drolysate.
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`CDNA Clone Isolation. The initial positive clone, 337, was
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`isolated from a hgtll (Young 3: Davis. 1983) human liver
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`' cDNA expression library kindly provided by Drs. S. L. C. Woo
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`and T. Chandra. This library was screened with (1) goat
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`inhibitor.
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`anti-human Cl inhibitor serum preabsorbed with hereditary
`angioneurotic edema serum (which was essentially devoid of
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`Cl inhibitor antigens) and (2) rabbit anti-human C1 inhibitor
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`serum preabsorbed with human serum albumin. Positive phage
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`were detected by using biotinylated second antibodies and
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`I25I—streptavidin. A29a, 313p, and RIOq (Figure 1c) Were
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`identified by hybridization with the 5’ £37 EcoRI fragment
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`and a pool of 96 17-base oligonucleotides encoding the hex—
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`apeptide Met-Leu-PheVaI-Glu—Pro (Harrison, 1983). hSq,
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`h2r, and Mr were obtained by again screening the library, this
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`time with the oligonucleotide dGTCAGCAGGGTCAGCC,
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`which had been identified at the 5' end of clone )tIOq on the
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`noncoding strand. Sequence analysis showed that ASq, Mr,
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`and Mr contain sequences at their 5’ ends (thick lines) that
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`correspond to sequences from elsewhere in the CT inhibitor
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`cDNA and that are in inverted repeat orientation to them.
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`This artifact of cDNA cloning occurs occasionally for full-
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`length cDNAs (Weaver et al., 1981). The cDNA clone pKll
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`was obtained from a pUC expression library (Helfman ct al.,
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`1983) synthesized from HepG2 (Knowles et al., 1980) poly-
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`(A)+ RNA and codes for a valine as residue 458.
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`DNA Sequencing Strategy. Fragments of the cDNA clones
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`shown in Figure lc were subcloned into pUC plasmids, and
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`sequence was obtained from linearized double-stranded DNA
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`by the dideoxy method (Sanger et al., I977). Most of the
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`cDNA was sequenced in both directions (Figure 1d): solid
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`amino acid sequence data was available for regions where it
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`was determined on only one strand.
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`Serpin Alignment. The alignment was obtained by visual
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`inspection of the mature protein sequences with due consid-
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`eration of already published comparisons (Petersen et at, 1979;
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`Hunt & Dayhoff, 1980; Carrel] et al., 1982; Doolittle, 1983,
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`1985; Hill et al., 1984; Hejgaard et al., 1985; Ragg. 1986).
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`Genomic DNA and Southern Blot Preparation. The
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`methods that were used for extracting genomic DNA from
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`peripheral blood samples of normals and HANE patients and
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`for preparing Southern blots have been described previously
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`(Bock et al.. 1985a).
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`Chromosomal localization. Southern blots containing
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`BomHI-digested DNA from 41 different human—mouse hy-
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`brids were hybridized with the human Cl inhibitor cDNA
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`probe and scored for the presence or absence of 4- and 6-kb
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`bands present in the human Ci inhibitor gene. The cell by-
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`brids were generated from 15 unrelated human cell lines and
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`4 mouse cell lines (Shows et al., 1982, 1984) and have been
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`characterized by chromosome analysis and mapped enzyme
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`markers and partly by mapped DNA probes (Shows et al.,
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`1978, 1982; Shows, i983).
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`RESULTS
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`Primary Structure. The