Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.     

The complete covalent structure of protein B. The third major protein degraded during germination of Bacillus megaterium spores

The complete covalent structure of Protein B, the third major protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with the specific B. megaterium spore protease into three peptides, residues 1 to 31 (B-III), 32 to 66 (B-I), and 67 to 96 (B-II). Cleavage of the intact protein with trypsin allowed isolation of the peptide encompassing residues 61 to 77 (T-11) as well as the COOH-terminal peptide, residues 94 to 96 (T-4). Cleavage of Peptide B-I with trypsin or chymotrypsin allowed isolation of peptides encompassing residues 53 to 60 (B-I-T-2) and residues 52 to 66 (B-I-C-4), respectively. Subtractive Edman degradation of Peptide T-4, automated sequenator analysis of Peptides B-I, B-II, T-11, B-I-T-2, and B-I-C-4, previously published partial sequence data on the intact B-protein and carboxypeptidase V digestion of the intact protein provided the data from which the following unique sequence was deduced: NH2-Ala-Lys-Gln-Thr-Asn-Lys-Thr-Ala-Ser-Gly-Thr-Ser-Thr-Gln-His-15 Val-Lys-Gln-Gln-Asp-Ala-Gln-Ala-Ser-Lys-Asn-Asn-Phe-Gly-Thr-30 Glu-Phe-Gly-Ser-Glu-Thr-Asn-Val-Gln-Glu-Val-Lys-Gln-Gln-Asn-45 Ala-Gln-Ala-Ala-Asn-Lys-Ser-Gln-Asn-Ala-Gln-Ala-Ser-Lys-60 Asn-Asn-Phe-Gly-Thr-Glu-Phe-Ala-Ser-Glu-Thr-Ser-Ala-Gln-Glu-75 Val-Arg-Gln-Gln-Asn-Ala-Gln-Ala-Gln-Lys-Lys-Asn-Gln-Asn-90 Ser-Gly-Lys-Tyr-Gln-Gly-COOH. The primary sequence of the B-protein contains a large internal duplication (residues 17 to 50 and 52 to 85), and shows significant sequence homology with the A- and C-proteins, the other major proteins degraded during B. megaterium spore germination.     

Determination of the complete amino acid sequence of bovine cardiac troponin C.

The amino acid sequence of bovine cardiac troponin C has been completely determined. The protein was cleaved by cyanogen bromide and the resulting peptides were isolated. All of the 161 residues of the protein could be accounted for in 12 cyanogen bromide peptides. Overlapping peptides were generated by tryptic digestion of citraconylated troponin C and isolation of the resulting five peptides. The primary structure of cardiac troponin C was elucidated by sequential manual Edman degradation of these peptides. It consists of four homologous regions, one of which probably has lost the ability to bind calcium ions. By comparing the amino acid sequence of cardiac troponin C with the sequence of skeletal troponin C, it was found that the mutation rate of the region that does not bind calcium is almost twice as high as the mutation rate of the three homologous regions that do bind calcium.     

Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).     

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 complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

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.     

Partial amino acid sequence of human plasma retinol-binding protein. Isolation and alignment of the five cyanogen bromide fragments and the amino acid sequences of four of the fragments

Studies are reported on the primary structure of human retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. The protein consists of a single polypeptide chain of 186-187 amino acids. RBP was cleaved by cyanogen bromide into five fragments, CB-I (27 residues), CB-11 (25 residues), CB-III (20 residues), CB-IV (15 residues), and CB-V (99-100 residues). The cyanogen bromide fragments were isolated, their compositions were determined, and they were aligned after studies that included the tryptic digestion of maleylated, reduced, and carboxymethylated RBP and subsequent enzymatic digestion of some of the resulting tryptic peptides. The amino acid sequences of four of the five cyanogen bromide fragments were determined, and the sequence of almost two-thirds of the NH2-terminal portion of the RBP molecule was determined as: H2N-GLU-Arg-Asp-Cys-Arg-Val-Ser-ser-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-Ser-Gly-Thr-Trp-Tyr-Ala-Met-Ala-Lys-Lys-Asp-Pro-Glu-Gly-Leu-Phe-Leu-Gln-Asp-Asx-Ile-Val-Ala-Glu-Phe-Ser-Val-Asx-Glx-Gly-Thr-Met-Ser-Ala-Thr-Ala-Gly-Lys-Arg-Val-Arg-Leu-Leu-Asn-Asn-Trp-Asp-Val-Cys-Ala-Asp-Met-Val-Gly-thr-Phe-Thr-Asp-Thr-Glu-Asp-Pro-Ala-Lys-Phe-Lys-Met-Lys-Tyr-Trp-Gly-Val-Ala-Ser-Phe-Leu-Gln-Lys-Gyl-Asn-Asp-Asx-His-Trp-Ile-Val-Asp-Thr-Asx-Thr-Tyr-Tyr-Ala-Val-Glu-Tyr-Cys-Ser-Arg---.     

Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.     

Selective dansylation of M protein within intact influenza virions.

Exposure of purified influenza virions to [14C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.     

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.     

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.     

A tryptic digestion fragment of nerve growth factor with nerve growth promoting activity

A peptide, isolated from the acid-insoluble portion of a tryptic digest of cyanogen bromide cleaved nerve growth factor, favors life maintenance of sensory target cells and promotes rapid neurite outgrowth from 7-day-old chick embryo sensory ganglia. The fragment has been identified as a 30 amino acid peptide consisting of two linear oligipeptides linked by a disulphide bridge and corresponding to residues 10–25 and 75–88 of the amino acid sequence of nerve growth factor. On a molar basis the fragment is about 100 times more effective than intact nerve growth factor. Other peptides isolated from the digest are biologically inactive.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).     

Amino acid sequence of Streptomyces metallo-proteinase inhibitor from Streptomyces nigrescens TK-23

Streptomyces Metallo-Proteinase Inhibitor (S-MPI) consists of 102 amino acid residues, including one methionine and two disulfide bridges. The complete amino acid sequence of S-MPI, including two disulfide bridges, was determined by sequencing of tryptic and chymotryptic peptides of two fragments obtained by cyanogen bromide cleavage followed by reduction and S-pyridylethylation of the protein. Incubation of the inhibitor with thermolysin slowly cleaved one peptide bond, Cys(64)-Val(65), which might be a reactive site of S-MPI.     

The primary structure of rat liver ribosomal protein L39

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.     

Covalent structural analysis of yeast inorganic pyrophosphatase.

The present paper describes the amino acid sequence analysis of the internal and COOH-terminal cyanogen bromide fragments of yeast inorganic pyrophosphatase (Sterner, R., Noyes, C., and Heinrikson, R.L. (1974) Biochemistry 13, 91-99). This information coupled with that derived from earlier structural studies of the enzyme (Sterner, R., AND Heinrikson, R.L. (1975) Arch. Biochem. Biophys. 165, 693-703) provides the complete covalent structure of the pyrophosphatase subunit. The majority of the sequence data was derived from automated Edman degradation of the intact cyanogen bromide fragments and the large tryptic peptides obtained from citraconylated derivates in which cleavages were restricted to arginyl residues. The structural determination was completed by analysis of tryptic and chymotryptic peptides from the decitraconylated fragments. The monomer peptide chain contains 285 amino acid residues and the molecular weight calculated from the sequence analysis is 32,042.     

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

Characterization of structural unit of phospholamban by amino acid sequencing and electrophoretic analysis

The partial amino acid sequence of phospholamban from canine cardiac sarcoplasmic reticulum was determined by sequence analysis of the peptides obtained from the protein cleaved by cyanogen bromide and with TPCK-trypsin. The sequence determined initiated with N alpha-acetylated methionine followed by 44 amino acid residues intervening two unidentified residues. This polypeptide would represent a structural unit (protomer) of phospholamban. Analysis of temperature-dependent conversion of phospholamban from 26 kDa to lower molecular weight form (6 kDa) suggested that phospholamban holoprotein is composed of five identical protomers.