Amino acid sequence of beta-galactosidase. XI. Peptide ordering procedures and the complete sequence.

The amino acid sequence of beta-galactosidase has been determined. The monomer contains 1,021 amino acid residues in a single polypeptide chain and has a molecular weight of 116,349. All 80 tryptic peptides as well as all 24 CNBr peptides have been isolated in pure form. Evidence is presented for the ordering of the CNBr peptides. The sequence determination was aided by analysis of cyanogen bromide peptides obtained from a polypeptide fragment produced by a lacZ termination mutant strain.     

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.     

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene

The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme.     

Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

The amino acid sequence of plastocyanin from Cucurbita pepo L. (vegetable marrow)

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A `tree' of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.     

The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.     

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequences of substrate-binding sites in chicken liver fatty acid synthase

The amino acid sequences of three essential regions of chicken liver fatty acid synthase have been determined: that around 4'-phosphopantetheine ("carrier" site), the substrate "loading" site containing serine, and a "waiting" site for the growing fatty acid containing cysteine. The amino acid sequence of the 4'-phosphopantetheine region was determined for the acetyl-, malonyl-, hydroxybutyryl-, and butyryl-enzyme with peptides obtained by hydrolysis of the enzyme with trypsin and Staphylococcus aureus (V8) protease. The sequence region around the essential serine was obtained for the acetyl- and malonyl-enzyme. The N-terminus of the tryptic peptide was blocked. However, the same sequence is obtained for the acetyl- and malonyl-peptide after S. aureus protease digestion, suggesting that the enzyme contains a single acyl transferase rather than two separate transacylases. The sequence around the cysteine was obtained by use of a radioactive iodoacetamide label. An unusual sequence of three serines adjacent to the cysteine was found. The strong similarities between peptides from different species for all three of the regions suggest that the multifunctional polypeptides from yeast and animals have evolved from the monofunctional enzymes of lower species.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

The amino-acid sequence of troponin C from frog skeletal muscle.

The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103--104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 152 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.     

TRYPTIC PEPTIDES FROM BOVINE WHITE MATTER PROTEOLIPIDS

Abstract— The amino acid composition of the fractions obtained after tryptic digestion of performic acid oxidized and non-oxidized white matter proteolipids was studied. The acid-soluble fraction from the tryptic digest represented between 25 and 30% of the starting material and was relatively enriched in hydrophilic amino acids and deficient in hydrophobic amino acids. The acid-soluble peptides were separated by high voltage paper electrophoresis, and the amino acid compositions of 16 peptides were determined; three additional peptides were obtained from the acid-soluble digest of the oxidized proteolipid. The sequence of 7 peptides including the N- and C-terminal peptides is reported. The results suggest that the protein is segregated into hydrophilic and hydrophobic regions and that small hydrophilic regions are separated by large hydrophobic areas.     

A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.

The amino acid sequence of the ferredoxin of Brassica napus was determined by using a Beckman 890C sequencer in combination with the characterization of peptides obtained by tryptic and chymotryptic digestion of the protein; some peptides were subdigested with thermolysin. The molecule consists of a single polypeptide chain of 96 amino acid residues and has an unblocked N-terminus. The primary structure shows considerable similarity with other plant-type ferredoxins.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

The primary structure of rabbit and rat prealbumin and a comparison with the tertiary structure of human prealbumin

The primary structures of rabbit and rat prealbumin have been determined. The amino acid sequence of rabbit prealbumin was determined by analyses of peptides obtained by trypsin and Staphylococcus aureus protease digestions. The rat prealbumin sequence was deduced by analyses of tryptic peptides as well as by nucleotide sequencing of cDNA clones. Both amino acid sequences contain 127 amino acid residues, the same as human prealbumin. Pairwise comparisons show that the three sequences are more than 80% identical. All three prealbumins were found to display significant sequence homology with human thyroxine-binding globulin. A comparison of the primary structures of the prealbumins with the tertiary structure of human prealbumin shows that amino acid replacements are preferentially located at the surface of the molecule and in the loops connecting the beta-strands. The locations of the replacements are discussed as regards the different molecular interactions in which prealbumin is involved.     

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.     

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.     

Studies on the high-sulphur proteins of reduced mohair. The isolation and amino acid sequence of protein scmkb-m1.2.

The complete amino acid sequence of mohair protein, SCMKB-M1.2 (97 residues), was determined. The protein was isolated from reduced and carboxymethylated mohair by chromatography on DEAE-cellulose phosphate. Peptides for sequence determination were obtained by digestion with trypsin, pepsin, chymotrypsin, thermolysin and papain, and were fractionated by DEAE-cellulose chromatography, paper chromatography and electrophoresis. The sequence of the peptides were determined by the Edman degradation method (by use of both the Beckman Sequence and a non-automatic procedure), and by partial acid hydrolysis. The protein is closely homologous to wool protein SCMKB-IIIB2, and also contains acetylated alanine as N-terminal amino acid.     

Primary structure of the inorganic pyrophosphatase from thermophilic bacterium PS-3

The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes.     

The amino acid sequence of plastocyanin from spinach. (Spinacia oleracea L.).

The amino acid sequence of spinach (Spinacia oleracea L.) plastocyanin was determined. It consists of a single polypeptide chain of 99 residues and has a sequence molecular weight of 10415. The sequence was determined by using a Beckman 890C automatic sequencer and by the dansyl--phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. Overlap through the two methionine residues was not shown. Sedimentation equilibrium in the ultracentrifuge gave a molecular weight for spinach plastocyanin of about 9000, in contrast with the value of 21000 reported previously by Katoh et al. (1962).