The complete amino acid sequence of adenylate kinase from baker's yeast

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP----MgADP + ADP) from baker's yeast has been determined. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid-phase sequencer or a gas-phase sequencer. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys-C and cyanogen bromide cleavages. The N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages. The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active-site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP:AMP phosphotransferase from beef heart mitochondria.     

Antigenic structure of adenylate kinase from porcine skeletal muscle. I. Three immunochemically active peptides obtained by cleavage with cyanogen bromide

Analysis of the quantitative precipitin reaction of adenylate kinase from porcine skeletal muscle with goat anti-adenylate kinase antiserum indicated that there are at least four antigenic determinants on the enzyme molecule. Porcine adenylate kinase was cleaved with cyanogen bromide, and four peptides were fractionated by ion-exchange chromatographies. Three fragments, CBb (2-56), CBfN (81-125), and CBfC (126-194), inhibited the quantitative precipitin reaction of intact adenylate kinase with goat antiserum. CBb, CBfN, and CBfC also inhibited the binding of 125I-labeled adenylate kinase to the specific antibody purified from goat antiserum. In both inhibition studies, the inhibitory activity of each fragment was extremely high, and reached 70% or more in the latter case. From these results and in view of the presence of the sequence -Glu-Glu-X-X'-Lys (or Arg)-Lys- in each immunochemically active fragment, we suggest that these fragments have similar antigenic determinants which are cross-reactive.     

Primary structure of mammalian ribosomal protein S6

Ribosomal protein S6 was isolated from rat liver ribosomes by reversed-phase high-performance liquid chromatography (HPLC) and subjected to cyanogen bromide and proteolytic cleavages. The cleavage fragments were resolved by HPLC and sequenced by automated Edman degradation. The overall amino acid sequence of S6 (249 residues) was determined by alignment of the overlapping sequences of selected cyanogen bromide, chymotryptic, tryptic, and clostripain cleavage fragments. The only protein found to exhibit close homology with the S6 sequence is yeast ribosomal protein S10 (61% sequence identity). Previously, characterized phosphopeptide derivatives of S6 containing phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent and protease-activated protein kinases originate from the carboxy-terminal region of S6 encompassing residues 233-249.     

Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase. Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced. Approximately 25-30% of the protein was sequenced in this manner. Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins. Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF. Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man. No ARF immunoreactivity was observed when Escherichia coli proteins were tested. Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant. Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein. ARF is shown to be myristylated at the amino terminus. The potential role of myristylation in cellular localization is discussed.     

Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase

The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.     

The primary structure of rabbit liver cytosolic serine hydroxymethyltransferase

The complete amino acid sequence of cytosolic serine hydroxymethyltransferase from rabbit liver was determined. The sequence was determined from analysis of peptides isolated from tryptic and cyanogen bromide cleavages of the enzyme. Special procedures were used to isolate and sequence the C-terminal and blocked N-terminal peptides. Each of the four identical subunits of the enzyme consists of 483 residues. The sequence could be easily aligned with the sequence of Escherichia coli serine hydroxymethyltransferase. The primary structural homology between the rabbit and E. coli enzymes is about 42%. The importance of the primary and predicted secondary structural homology between the two enzymes is discussed.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase

Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine. Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme. Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.     

Structural and catalytic characteristics of Escherichia coli adenylate kinase

The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.     

Production and Isolation of Levan by Use of Levansucrase Immobilized on the Ceramic Support SM-10

The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethyl ated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments.

RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.     

Complete amino acid sequence of yeast thioltransferase (glutaredoxin)

The amino acid sequence of a thioltransferase isolated from Saccharomyces cerevisiae was determined. The protein was cleaved by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino acid residues with two cysteines. Yeast thioltransferase showed 51% structural similarity to pig liver thioltransferase and 34% to E. coli glutaredoxin.     

Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides

As a part of the elucidation of the complete amino acid sequence of human phosphoglycerate kinase, 46 tryptic peptides, ranging in length from 1 to 26 residues, were isolated and characterized from the reduced and S-carboxymethylated enzyme. The isolated peptides were subjected to sequence analysis by the modified dansyl-Edman degradation procedure and automated Edman degradation technique. The results, together with the data on cyanogen bromide peptides and two additional tryptic peptides from cyanogen bromide peptides reported in the accompanying paper, established the complete amino acid sequence of human erythrocyte phosphoglycerate kinase.     

Major proteins of the Escherichia coli outer cell envelope membrane. Sequence of the cyanogen bromide fragments of protein I from Escherichia coli B/r.

The sequence of the cyanogen bromide fragments of one of the major outer membrane proteins of E. coli B/r has been established with the aim of elucidating the primary structure of this protein. Separation of all fragments on one molecular sieve column was achieved upon citraconylation of these fragments. Overlapping peptides were obtained by digestion of the protein, or a cyanogen bromide fragment arising from incomplete cleavage, with trypsin or Staphylococcus aureus protease.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic alpha-amylase

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary alpha-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian alpha-amylases.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.     

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.     

Guanylate kinase from Saccharomyces cerevisiae. Isolation and characterization, crystallization and preliminary X-ray analysis, amino acid sequence and comparison with adenylate kinases

This paper describes a large-scale purification of guanylate kinase (ATP + GMP in equilibrium ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X-ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences. 1. Guanylate kinase was purified in five steps to homogeneity: crude extract, ion-exchange chromatography, affinity chromatography and gel filtration twice. 2. The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 x 200 x 150 microns 3. Preliminary X-ray results are given. 3. The final sequence shows 186 amino acids (Mr = 20,548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N-terminal blocking group was identified by 1H-NMR spectroscopy. 4. Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Porcine pancreatic α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.     

Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.