Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia coli.

The sequence of the gene coding for GTP cyclohydrolase I of Escherichia coli and of the adjacent regions was determined. The open reading frame contains 669 nucleotides. The deduced amino-acid sequence represents a protein consisting of 223 amino-acid residues with a molecular mass of 24,873 Da. Partial amino-acid sequences of the N-terminal region and of 5 peptides obtained by trypsin and BrCN cleavage were determined by Edman degradation and were in full agreement with the sequence deduced from the nucleotide sequence. The starting methionine is removed by posttranslational modification. The protein shows extensive homology to the recently reported GTP cyclohydrolase from rats.     

Complete amino-acid sequence of bovine seminal ribonuclease, a dimeric protein from seminal plasma

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.     

Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.     

Pig beta-lactoglobulin I (Sus scrofa domestica, Artiodactyla). The primary structure of the major component

beta-Lactoglobulins from pooled milk (Sus scrofa domestica) are isolated and characterized. The complete primary structure of the major beta-lactoglobulin component I is presented. The amino-acid sequence was elucidated by automated Edman degradation of tryptic peptides and cyanogen bromide cleavage products in a liquid phase sequencer. The tryptic and cyanogen bromide peptides were separated by reverse-phase (RP-2) or size exclusion (TSK 2000 SW) high performance liquid chromatography. Pig beta-lactoglobulin is composed of only 159 amino acids in contrast to other beta-lactoglobulins which contain 162 or 166 amino acids. Sequence alignment with previously sequenced beta-lactoglobulins was obtained by introducing two gaps at positions 115 and 151-152. Thus bovine beta-lactoglobulin A reveals 62 amino-acid substitutions. The phylogenetic distance from horse beta-lactoglobulin I and II is indicated by 49.4% and 62% amino-acid exchanges, respectively. Pig beta-lactoglobulin is a mixture of two chains with Gln or Thr at position 119. The free thiol group is localized at position 59. The structural and functional aspects of beta-lactoglobulins and its role in vitamin A (retinol) transport are discussed.     

A new strategy for primary structure determination of proteins: application to bovine beta-casein

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C-terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS-PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N-terminal sequence of peptide (i-1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine beta-casein (209 residues) which was completely resequenced with only 239 Edman cycles.     

Amino acid sequence of the Pseudomonas putida cytochrome P-450. I. Sequences of tryptic and clostripain peptides

In order to elucidate the complete amino acid sequence of Pseudomonas putida cytochrome P-450, tryptic digestion was performed on the S-carboxymethylated enzyme. Although cleavage did not occur at every lysyl and arginyl bond, 31 tryptic peptides ranging in size from 1 to 55 residues were isolated. These were sequenced by manual Edman degradation and carboxypeptidase digestion. Overlaps of some od these tryptic peptides were obtained by data obtained from partial Edman degradation and amino acid composition of the clostripain cleavage products. These results, together with data from the cyanogen bromide and acid cleavage peptides reported in the accompanying paper, established the complete amino acid sequence of P. putida cytochrome P-450.     

The primary structure of the psychrophilic lactate dehydrogenase from Bacillus psychrosaccharolyticus

L-lactate dehydrogenase of the psychrophilic bacterium B. psychrosaccharolyticus was isolated by a three-step procedure and its total amino-acid sequence determined by automated Edman degradation. The protein consists of 318 amino-acid residues and its calculated molecular mass is 35,254 Da. Most of the primary structure could be established by sequencing large peptide fragments obtained by chemical cleavages, namely with BNPS-skatole and with CNBr. Further fragmentations of two tryptophan peptides with the endoproteinase Lys-C and with diluted HCl resulted in shorter overlapping peptides, the analysis of which completed the sequence. The C-terminal sequence Glu-Gln was established by carboxypeptidase A experiments and was then verified by the analysis of short C-terminal tryptic and chymotryptic peptides. The first lactate dehydrogenase sequenced so far of a psychrophilic bacillus shows sequence homologies between 60% and 75% to the enzymes from the mesophilic B. megaterium and B. subtilis and the thermophilic B. stearothermophilus, B. caldolyticus and B. caldotenax. Within the 50 N-terminal residues, three additional sequences could be included in our comparisons. In this part of the molecule, sequence homologies between 56% and 74% were calculated.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.     

Amino acid sequence of a unique neuronal protein: rat olfactory marker protein

A neuron-specific protein, the olfactory marker protein (OMP), has been sequenced. This was achieved by gas phase sequencing of peptides isolated by HPLC following chemical and enzymatic cleavages of the intact rat protein. The amino terminus of the intact protein is acetylated. This has been determined by fast atom bombardment mass spectrometry of the amino terminal dodecapeptide isolated following BrCN cleavage of the OMP. Comparison of the sequence reported here with over 3000 protein sequences stored in the NBRF protein data base indicates no significant homology with any previously sequenced protein. This, coupled with the occurrence of OMP only in mature olfactory neurons of many vertebrate species, suggests that this protein has a olfactory neurons of many vertebrate species, suggests that this protein has a unique function in the metabolism of these neurons.     

The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins

beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse beta-lactoglobulin II is unique in that it contains 166 amino acids. The additional four amino acids represent an insertion between positions 116 and 117 of other beta-lactoglobulins so far sequenced, including horse beta-lactoglobulin I. Sequence comparison of beta-lactoglobulins I and II from horse colostrum reveals 48 amino acid substitutions (30%). Such a diversity between members of the beta-lactoglobulin gene family has not been encountered before. Sequence comparison with bovine beta-lactoglobulin A shows 85 amino acid replacements accounting for 53% of the residues. The structural homology with human retinol-binding protein may reveal similar biological functions and clues to the origin of milk proteins.     

The amino-acid sequence of rat Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase isolated from rat liver was determined. The enzyme was reduced, carboxymethylated and fragmented by treatment with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by gel filtration or high performance liquid chromatography and sequenced by automated Edman degradation. The total sequence of 153 amino-acid residues per subunit was reconstructed from overlapping peptides. Rat Cu-Zn superoxide dismutase proved to be closely related to the corresponding sequences of other mammals in having more than 80% identical amino-acid residues in homologous position and an acetylated N-terminus. Comparison of the rat Cu-Zn superoxide dismutase structure with those of other species suggests a similar phylogenetic distance between rat, man, pig, cattle and horse and a rapid molecular divergence during vertebrate development compared to earlier evolutionary periods.     

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.     

Studies on the structure of rabbit muscle aldolase. 3. Primary structure of the BrCN peptide containing the active site

The four peptide segments obtained from rabbit muscle aldolase by cleavage with BrCN and separation with gel-filtration chromatography (1) have been redesignated according to their positions in the molecule, N-A-B-C. The primary structure of segment A, containing 66 amino acid residues, including the Schiff base-forming lysine at the active site, has been elucidated by isolation and sequence analyses of the proteolytic subfragments. Preliminary separation of tryptic peptides containing 7–25 residues was achieved by chromatography on Sephadex G-25 which facilitated subsequent purification. For the study of the tryptic peptide of 25 residues further fragmentation with pepsin then subtilisin (Nagarse) was employed. Edman degradation directly after subtilisin cleavage of a peptide was found useful in avoiding deamidation of a glutamine NH₂-terminus newly formed in the proteolysis. The sequence of 90 amino acids in the center region of the polypeptide chain of rabbit muscle aldolase has now been established.     

Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats

The amino acid sequence of human plasma prekallikrein was determined by a combination of automated Edman degradation and cDNA sequencing techniques. Human plasma prekallikrein was fragmented with cyanogen bromide, and 13 homogeneous peptides were isolated and sequenced. Cyanogen bromide peptides containing carbohydrate were further digested with trypsin, and the peptides containing carbohydrate were isolated and sequenced. Five asparagine-linked carbohydrate attachment sites were identified. The sequence determined by Edman degradation was aligned with the amino acid sequence predicted from cDNAs isolated from a lambda gt11 expression library. This library contained cDNA inserts prepared from human liver poly(A) RNA. Analysis of the cDNA indicated that human plasma prekallikrein is synthesized as a precursor with a signal peptide of 19 amino acids. The mature form of the protein that circulates in blood is a single-chain polypeptide of 619 amino acids. Plasma prekallikrein is converted to plasma kallikrein by factor XIIa by the cleavage of an internal Arg-Ile bond. Plasma kallikrein is composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), and these 2 chains are held together by a disulfide bond. The heavy chain of plasma kallikrein originates from the amino-terminal end of the zymogen and is composed of 4 tandem repeats that are 90 or 91 amino acid residues in length. These repeat sequences are also homologous to those in human factor XI. The light chain of plasma kallikrein contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.     

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.     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene

The amino-acid sequence of the single-stranded DNA-binding protein of bacteriophage Pf1 and the nucleotide sequence of the corresponding gene have been determined. The protein has 144 amino acids and a molecular weight of 15 400; the gene consists of 435 nucleotides. The amino-acid sequence was determined by Edman degradation, carboxypeptidase A, B, and P digestion of intact protein and of peptides derived by chymotrypsin, Staphylococcus aureus V8 protease, and trypsin digestion. The nucleotide sequence was determined by the dideoxy method after random cloning of fragments of Pf1 DNA into M13. No sequence homology could be established between the amino-acid sequence of the DNA-binding protein of Pseudomonas aeruginosa-specific bacteriophage Pf1 and bacteriophage fd of Escherichia coli.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit

The amino acid sequence was determined for equine lutropin beta (eLH beta). Large fragments were derived from reduced, carboxymethylated eLH beta by digestion with Staphylococcus aureus V8 protease, by cyanogen bromide cleavage, and by cleavage of acid-labile Asp-Pro bonds. The fragments were purified by gel filtration and high performance liquid chromatography (HPLC). The fragments were sequenced by automated Edman degradation to establish the primary structure of eLH beta. Some peptides were further digested with chymotrypsin and the resulting peptides purified by HPLC. In addition to sequencing by automated Edman degradation, these were also sequenced by the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure which enabled us to directly identify glycosylated amino acids. The eLH beta subunit is a glycoprotein of 149 amino acids containing both N- and O-linked oligosaccharides. It possesses a COOH-terminal extension similar to that seen in human chorionic gonadotropin. Carboxypeptidase Y digestions suggest that the COOH terminus is blocked by glycosylation. Interestingly, the amino acid sequence of eLH beta is identical to that of equine chorionic gonadotropin beta (Sugino, H., Bousfield, G. R., Moore, W. T., and Ward, D. N. (1987)J. Biol. Chem. 262, 8603-8609).     

The complete primary structure of alpha-lactalbumin isolated from pig (Sus scrofa) milk

The complete amino-acid sequence of pig alpha-lactalbumin has been determined. It was obtained by microsequencing of the native protein and the peptides derived after tryptic or cyanogen bromide cleavage. The tryptic peptides were separated by a rapid microbore HPLC method. Pig alpha-lactalbumin is 122 amino acids long and differs from the bovine homologue by 26 exchanged residues. Of the two prolines present in bovine alpha-lactalbumin, one has been deleted in the pig structure. All previously sequenced alpha-lactalbumins have shown glutamic acid at position 49, which is known to be the active site in the homologous lysozyme c structure. This residue is replaced by phenylalanine in pig alpha-lactalbumin indicating that the pig protein is the first alpha-lactalbumin with complete loss of all lysozyme functional residues.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).