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Bovine pancreatic deoxyribonuclease A. Isolation of cyanogen bromide peptides; complete covalent structure of the polypeptide chain.

Abstract The following sequence and the pairing of the half-cystine residues has been derived for bovine pancreatic deoxyribonuclease A. [see PDF for sequence] The sequence permits the following assignments of residues known to have special properties. The carbohydrate moiety is attached to Asn18. The crucial histidine residue subject to alkylation by iodoacetate is His131. The site of nitration when the enzyme is inactivated by tetranitromethane is Tyr62; the secondary site of nitration is Tyr73. The essential disulfide bond links residues 170 and 206.

Primary Sequence of U-1 Nuclear Ribonucleic Acid of Novikoff Hepatoma Ascites Cells

The U-1 RNA is one of the low molecular weight nuclear RNAs of Novikoff hepatoma ascites cells that is specifically localized to the extranucleolar portion of the nucleus. After the RNA was purified by polyacrylamide gel electrophoresis, it was subjected to various enzymatic digestion procedures for determination of its primary nucleotide sequence. The U-1 RNA contains 171 nucleotides. Its 5′ end is an alkali-stable tetranucleotide containing N2,2,7-trimethyl guanylic acid. The primary sequence of U-1 RNA is: (m32,2,7G, Am, Um) [see PDF for sequence]

California Sea Lion Myoglobin: COMPLETE COVALENT STRUCTURE OF THE POLYPEPTIDE CHAIN

The following sequence has been derived for the myoglobin of the California sea lion: [see PDF for sequence]. The sequence differs from that of harbor seal myoglobin at 17 positions and from that of sperm whale myoglobin at 27 positions. By comparison with these and other myoglobins it appears that those residues that in the sperm whale protein make direct contact with the heme or are directed inward are most closely conserved, followed by residues in the nonhelical regions, lastly followed by those in helical segments. The methodology made use of cleavages by trypsin, thermolysin, chymotrypsin, or cyanogen bromide, with or without exhaustive carboxymethylation of the apomyoglobin substrate. Sequence were determined by Edman degradation, both manual and automated, with frequent use of aminopeptidase M and carboxypeptidase C.

Structural Analysis of Ribosomal 28 S Ribonucleic Acid of Novikoff Hepatoma Cells: PRIMARY SEQUENCE OF THE B3-9 SUBCOMPONENT

The primary sequence of nucleotides was determined for the 99 nucleotide B3-9 subcomponent of 28 S rRNA of Novikoff hepatoma ascites cells; this fragment was purified from the 500 nucleotides GC-rich B3 fragment that is produced by partial cleavage of 28 S rRNA with T1 RNAase. This unique sequence, which is the longest defined thus far for 28 S rRNA, is: see PDF for sequence The only Ap residue is in the 5′-terminal end. The italicized polypyrimidine sequences which have chain lengths of 17, 14, and 21, respectively, are unique markers since they appear only once in 28 S rRNA ot its nucleolar precursors.

Structure of the Carbohydrate Units of IgE Immunoglobulin: II. SEQUENCE OF THE SIALIC ACID-CONTAINING GLYCOPEPTIDES

Abstract In the preceding paper (Baenziger, J., Kornfeld, S., and Kochwa, S. (1974) J. Biol. Chem. 249, 1889) it was shown that an IgE myeloma protein has 4 oligosaccharide units per heavy chain, 1 of which contains only mannose and N-acetylglucosamine while the other 3 contain sialic acid, fucose, galactose, mannose, and N-acetylglucosamine in the ratio of 1 to 2:1:2:3:4. The data in this paper establish the structure of the three sialic acid-containing glycopeptides, designated B-1, B-2, and B-3. Glycopeptides B-2 and B-3 are branched structures containing two nonreducing termini with the sequence sialic acid (α2,6)/→ Gal (β1,4)/→ GlcNAc (β1,2)/→ Man (α)/→, while B-1 has one nonreducing terminus with the above structure and another with the sequence Gal (β1,4)/→ GlcNAc (β1,2)/→ Man (α)/→. The core of all three B-glycopeptides is identical and has the sequence: [see PDF for sequence] The branch of B-1 terminating in sialic acid originates from position 3 of the β-linked mannose in the core while the branch terminating in galactose originates from position 6 of this mannose.

Blood Group A Active Glycolipids of Hog Gastric Mucosa: ISOLATION AND PARTIAL CHARACTERIZATION

Abstract Glycolipids with blood group A activity were purified from hog stomach mucosa powder. The active lipids were isolated by chloroform-methanol extraction, partition with aqueous KCl followed by precipitation of the polar lipids with acetone. Then the A active glycolipids were purified by DEAE-cellulose and Florisil column chromatography. Final purification of the A active compounds was achieved by sequential preparative thin layer chromatography in two solvent systems. The homogeneity of the purified fractions was confirmed by thin layer chromatography in neutral, acidic, and basic solvent systems, thin layer chromatography of the acetylated derivatives of the purified fractions and by osmium-catalyzed periodate oxidation followed by paper chromatography of the released oligosaccharide chains. Two similar but distinct A active glycolipid fractions (L and U) were isolated. Fraction U contained high amounts of long chain hydroxy fatty acids, while Fraction L was rich in C16-C18 fatty acids. Sphingenine and heptadecasphinganine were the major long chain bases found in both fractions. The carbohydrate composition of Fractions L and U was identical and was found to be (in moles per 1 mole of glucose): galactose, 3.04; fucose, 1.09, N-acetylglucosamine, 0.99, N-acetyl-galactosamine, 0.93. Partial acid hydrolysis, mild acid hydrolysis, enzymatic digestion, and immunological assays established the following carbohydrate sequence for the A active glycosphingolipid isolated from hog stomach mucosa powder: [see PDF for sequence]

Identification of Aspartic Acid 52 as the Point of Attachment of an Affinity Label in Hen Egg White Lysozyme

Abstract Hen egg white lysozyme, fully inactivated by the affinity-labeling reagent, the 2',3'-epoxypropyl β-glycoside of di-(N-acetyl-d-glucosamine), contained 1 mole of the label per mole of protein. Total enzymatic digestion of the reduced and carboxymethylated affinity-labeled enzyme afforded, as a major radioactive product, a compound composed solely of aspartic acid and glucosamine at the molar ratio of 1:2. Peptic digestion afforded a single radioactive peptide corresponding to the sequence [see PDF for sequence] in the enzyme. Digestion of this peptide with Clostridium histolyticum aminopeptidase gave a radioactive pentapeptide the composition, partial structure, and other properties of which corresponded to the sequence [see PDF for sequence] in hen egg white lysozyme, and which contained 2 moles of glucosamine per mole of peptide. Our findings show that the affinity label is covalently bound to the enzyme via the β-carboxyl group of Asp 52 and are in accord with our conclusions from immunochemical (Maron, E., Eshdat, Y., and Sharon, N. (1972) Biochim. Biophys. Acta 278, 243) and x-ray crystallographic (Moult, J., Eshdat, Y., and Sharon, N. (1973) J. Mol. Biol. 75, 1) studies of the affinity-labeled hen egg white lysozyme.

The Primary Structure of the Clostridium thermosaccharolyticum Ferredoxin, a Heat-stable Ferredoxin

Abstract The amino acid sequence of Clostridium thermosaccharolyticum ferredoxin was determined to be:[see PDF for sequence]. Compared to the heat-stable ferredoxin from Clostridium tartarivorum, only two differences in the sequence were noted. Glutamine-31 and glutamine-44 in C. tartarivorum ferredoxin were replaced by glutamic acid in the C. thermosaccharolyticum ferredoxin. These changes increase the heat stability of the ferredoxin, since the C. thermosaccharolyticum ferredoxin is the most heat stable of all the ferredoxins isolated thus far. The structure of the heat-stable ferredoxin is discussed and compared with the three-dimensional structure of the Micrococcus aerogenes ferredoxin, a non-heat-stable ferredoxin which has just been completed (Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987–3996).

The Primary Structure of Peptostreptococcus elsdenii Flavodoxin

The amino acid sequence and the studies which led to the complete sequence determination of the Peptostreptococcus elsdenii flavodoxin are presented in this report. The sequence of the flavodoxin is of interest since it is the first flavoprotein whose sequence has been determined completely. The P. elsdenii flavodoxin is a FMN protein containing 137 amino acids. This single polypeptide chain protein was shown to have the sequence [see PDF for sequence]

The Nucleotide Sequence of the GGG-specific Glycine Transfer Ribonucleic Acid of Escherichia coli and of Salmonella typhimurium

Abstract The primary nucleotide sequence of tRNAggggly (glycine tRNA specific for the codon GGG) from Escherichia coli and Salmonella typhimurium has been determined. The E. coli tRNA sequence is [see PDF for sequence] where U at position 8 is most likely 4-thiouracil. The sequence of the S. typhimurium tRNAggggly is identical with that of E. coli except that no evidence for the modification of U at position 8 has been found. In addition, a portion of the S. typhimurium tRNAggggly has a 2'-O-methyl modification of the G at position 17. Some unusual features of the tRNA structure are discussed.

The Nucleotide Sequence of Saccharomyces cerevisiae 5.8 S Ribosomal Ribonucleic Acid

Abstract The nucleotide sequence of Saccharomyces cerevisiae 5.8 S ribosomal RNA (also known as the 7 S or 1RNA species) has been determined to be [see PDF for sequence]

Cocoonase: V. STRUCTURAL STUDIES ON AN INSECT SERINE PROTEASE

Abstract Prococoonases and cocoonases from three species of silkmoths (Antheraea pernyi, Antheraea polyphemus, and Antheraea mylitta) have been purified and compared to each other and to vertebrate trypsins by tryptic peptide mapping, amino acid and carbohydrate composition, and partial amino acid sequence. Comparison of tryptic peptide maps of two cocoonases with bovine trypsin shows 50% coincidence of peptides. The prococoonases from A. pernyi and A. polyphemus are glycoproteins containing 6 moles of mannose and 2 moles of glucosamine. Proteolytic activation of prococoonase to cocoonase does not involve a loss of any of this carbohydrate. Prococoonase from A. mylitta contains no carbohydrate. Sequence analyses of the NH2 and COOH termini of several cocoonases and A. mylitta prococoonase and the entire active site tryptic peptide from A. polyphemus cocoonase reveals 70% identity with bovine trypsin. The partial structure of A. polyphemus cocoonase is [see PDF for sequence] The active site serine is indicated by the box.

Oligosaccharides Produced by Acetolysis of Blood Group Active (A + H)sulfated Glycoproteins from Hog Gastric Mucin

Abstract Blood group (A + H) sulfated glycoproteins containing oligosaccharides of 14 sugar residues have been purified from hog stomach mucosa, and the structure of the carbohydrate chains was investigated. Penta-, tetra-, tri-, and disaccharides were isolated from the products of acetolysis of this material, and their structures were studied by chemical, enzymatic, and immunological methods. Periodate oxidation of a reduced sulfated disaccharide composed of N-acetylglucosamine gave predominantly erythritol, indicating 1 → 4 linkage between the two glucosamine residues. Galactose and N-acetylglucosamine were obtained from the treatment of trisaccharide with β-galactosidase. Methylation analysis of the trisaccharide revealed equal amounts of acetates of 2,3,4,6-tetra-O-methylgalactitol and 2,4,6-tri-O-methylgalactitol in addition to 3,6-di-O-methyl-N-methylglucosaminitol. This is consistent with the structure Gal (β1-3)/→ Gal (β1-4)/→ GlcNAc. Blood group H-active monofucosyl penta- and tetrasaccharides were resistant to β-galactosidase, but were cleaved to the monosaccharides by a mixture of α1,2-l-fucosidase, β-galactosidase, and β-N-acetylglucosaminidase. Analysis of the methylated pentasaccharide revealed the presence of acetates of 2,3,4-, 2,3,6-, and 3,4,6-tri-O-methylgalactitol in approximately equal amounts and, in addition, 2,3,4-tri-O-methylfucitol and 3,6-di-O-methyl-N-methylglucosaminitol. These data are consistent with the structure Fuc (α1-2)/→ Gal (β1-4)/→ GlcNAc (β1-4)/→ Gal (β1-6)/→ Gal. Methylation analyses of one of the two tetrasaccharides showed acetates of 2,3,4-tri-O-methylfucitol, 3,4,6-, 2,3,6-tri-O-methylgalactitol, and 3,6-di-O-methyl-N-methylglucosaminitol, and for the other acetates of 2,3,4-tri-O-methylfucitol, 3,4,6-, 2,3,6-tri-O-methylgalactitol, and 4,6-di-O-methyl-N-methylglucosaminitol. Those data are consistent with the structures Fuc (α1-2)/→ Gal (β1-4)/→ GlcNAc (β1-4)/→ Gal and Fuc (α1-2)/→ Gal (β1-3)/→ GlcNAc (β1-4)/→ Gal. The suggested structure of blood group (A + H) -sulfated glycoprotein presented below is further substantiated. [see PDF for sequence]

On the Subunit Structure of Yeast Inorganic Pyrophosphatase

Abstract Evidence based upon a variety of experimental approaches is in accord with the conclusion that native yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) comprises two identical subunits approximately 32,000 in molecular weight with threonine and valine the NH2- and COOH-terminal residues, respectively. Gel filtration of reduced and carboxymethylated pyrophosphatase on a column of 6% agarose in 6 m guanidine hydrochloride revealed a single protein component of molecular weight 33,000; a value of 35,500 was obtained after exhaustive succinylation of the alkylated enzyme. The molecular weight thus derived for S-carboxymethylated pyrophosphatase was in close agreement with the values calculated from polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid compositional data which were, respectively, 33,000 and 30,250. Threonine was identified as the sole NH2-terminal residue by the dimethylaminonaphthalene-5-sulfonyl chloride end group procedure. Hydrolysis of alkylated pyrophosphatase by carboxypeptidase A was attended by the rapid release of COOH-terminal valine in quantities stoichiometric with the amount of monomeric protein present. Peptide maps of trypsin digests from S-carboxymethylated enzyme revealed a total of 30 peptides, five of which gave a positive test for arginine and three of which stained positively for tryptophan. These results are in accord with what would be expected on the basis of the lysine, arginine, and tryptophan content of the enzyme subunit. Finally, the partial sequence of NH2-terminal amino acid residues presented below was elucidated by Edman degradation with the aid of automated sequencing equipment. [see PDF for sequence] Yields of the phenylthiohydantoin amino acid derivatives obtained after each cycle of the degradative procedure were consistent with the quantity of monomeric protein subjected to analysis.

The Primary Structure of Epidermal Growth Factor

The complete amino acid sequence of epidermal growth factor (EGF) has been established through the use of automated Edman degradation and standard enzymatic and chemical techniques. The location of the half-cystinyl residues was facilitated by the use of the S-[14C]aminoethyl derivative of EGF. The sequence is: [see PDF for sequence]. The calculated molecular weight of the 53-residue polypeptide is 6045, a value that is in agreement with the molecular weight of 6400 established by physical measurements. The peptide is acidic and the 6 half-cystines exist in disulfide linkage. Four arginine residues are located in the COOH-terminal portion of the molecule. The six COOH-terminal amino acids are not necessary for biological activity and EGF1–47 has activity identical with that of the 53-residue native molecule.

Primary Structure of Escherichia coli Glutamine Synthetase: III. CHEMICAL EVIDENCE FOR SUBUNIT IDENTITY; AMINO ACID SEQUENCES AT THE NH2 AND COOH TERMINI

Abstract Evidence that the glutamine synthetase dodecamer from Escherichia coli is comprised of 12 identical subunits of molecular weight 49,000 has been obtained by end group analysis. Application of the carbamylation procedure to the NH2-terminal analysis revealed only serine. The yield was 98% of that expected from a polypeptide of the size proposed for the monomeric species. The following partial sequence of residues at the NH2 terminus of the molecule was elucidated with the aid of automated sequencing equipment: [see PDF for sequence] Hydrolysis of reduced and carboxymethylated glutamine synthetase by carboxypeptidase A yielded quantitative evidence for the following COOH-terminal sequence: -Leu-Tyr-Tyr-Ser-Val-COOH One mole of valine was released per mole of protein, assuming the subunit molecular weight to be 49,000.

COOH-terminal Amino Acid Sequence of Histidinol Dehydrogenase from a Salmonella typhimurium Mutant

Abstract A spontaneous prototrophic mutation in the hisD gene of Salmonella typhimurium, hisD2352, alters the COOH-terminal amino acid sequence of the hisD gene product, histidinol dehydrogenase. The wild type sequence, [see PDF for sequence] has been replaced by the sequence -Lys-Ala-Ser-Leu-Thr in the hisD2352 dehydrogenase. The permuted amino acid sequence may be explained if hisD2352 is a two-base deletion near the end of the hisD gene. A unique nucleotide sequence may be written for this region which indicates that the normal termination signal of the hisD gene is UAA or UGA. This single termination codon is followed by a cytosine residue, which constitutes part of an intercistronic region between the hisD and hisC genes.

Nucleotide Sequence of 4.5 S Ribonucleic Acidi of Novikoff Hepatoma Cell Nuclei

Abstract The primary sequence of nucleotides has been defined for 4.5 S RNAi, which is one type of low molecular weight nuclear RNA of Novikoff hepatoma ascites cells. This RNA is the first unique nuclear species of mammalian RNA to be sequenced. It is specifically localized to the extranucleolar portion of the nucleus and is not a precursor of tRNA. Highly labeled 4.5 S RNAi was obtained from cells incubated with [32P]orthophosphate in vitro. The RNA was purified by successive gel electrophoresis and chromatography on DEAE-Sephadex. The primary sequence of nucleotides of this RNA shows that it has a purine-rich 5' end and a pyrimidine-rich 3' end: [see PDF for sequence].

The Isolation and Primary Structure of a Peptide Containing the Oxidation-Reduction Active Cystine of Escherichia coli Lipoamide Dehydrogenase

An 11-residue peptic peptide from the catalytic center of Escherichia coli lipoamide dehydrogenase has been isolated and sequenced. This peptide contains the oxidation-reduction active disulfide of the enzyme which functions in concert with the flavin during catalysis. The peptide was isolated from a digest of a derivative in which the free sulfhydryls of the enzyme were substituted with the colored maleimide N-(4-dimethylamino-3,5-dinitrophenyl)maleimide. The sequence of the peptide was determined by a subtractive Edman degradation to be: [see PDF for sequence] This work suggests that the oxidation-reduction active disulfide of E. coli lipoamide dehydrogenase is contained in a hydrophobic pocket at the catalytic center of the enzyme, consistent with the binding of the nonpolar substrate dihydrolipoamide. Evidence is also presented that the two subunits of the enzyme are identical.

The Isolation and Primary Structure of a Peptide Containing the Oxidation-Reduction Active Cystine of Escherichia coli Thioredoxin Reductase

Abstract An octapeptide from Escherichia coli thioredoxin reductase has been isolated and sequenced; it contains the cystine previously shown to take part in electron transfer. The peptide was isolated from a peptic digest of a derivative in which the free sulfhydryls of the enzyme had been reacted with the colored maleimide, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide. The sequence of this peptide as determined by subtractive Edman degradation was: [see PDF for sequence] The minimum molecular weight based on amino acid analysis and flavin content has been redetermined on highly purified enzyme to be 36,500 per FAD or 73,000 per mole of enzyme. The total half-cystine content was found to be five per FAD, indicating three cysteines and one cystine per FAD.