The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.     

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.     

Amino acid sequences of portions of the alpha and beta chains of bovine fibrinogen.

The N-terminal portions of the Aα and Bβ chains of bovine fibrinogen (CNBr Aα and Bβ), each of which contains an ArgGly bond that is hydrolyzed by thrombin, have been isolated by cyanogen bromide cleavage of fibrinogen and column chromatography of the resulting material. These peptides were digested with thrombin, releasing fibrinopeptide A and GlyProArg from CNBr Aα, and fibrinopeptide B from CNBr Bβ. The C-terminal peptides produced by digestion with thrombin (CNBr α and CNBr β) were purified, and the amino acid sequences of portions of these peptides (30 residues from the N-terminus of CNBr α and 32 residues from the N-terminus of CNBr β) were determined with an automatic sequenator using the Edman degradation.     

The primary structure of actin from rabbit skeletal muscle. Completion and analysis of the amino acid sequence.

Actin is the principal constituent of the thin filaments of muscle, and in order to provide information basic to understanding the molecular basis of actin function we have studied its amino acid sequence. The isolation, compositions, and sequences of cyanogen bromide peptides, ranging in size from 3 to 44 residues, have previously been reported (ELZINGA, M. (1971) Biochemistry 10, 224-229, and other papers in the present series). The peptides have been aligned by isolation and characterization of tryptic peptides that contain methionine. The isolation of one of the CNBr peptides (CB-14) was complicated by the presence of a Met-Thr bond that was only partially split under standard conditions for cyanogen bromide cleavage in formic acid. In this paper conditions are described for increasing the cleavage at this bond. CB-14 is a tetrapeptide, Thr-Gln-Ile-Hse, and this sequence completes the characterization of the actin cyanogen bromide peptides. Finally, the position of CB-14 in the actin sequence as residues 120 to 123 was established by isolation of a chymotryptic overlap peptide. The complete sequence of the 374 residues of actin is presented.     

Amino Acid Sequence of Cyanogen Bromide Fragment CB I from Ala Chain of Ricin D

The amino acid sequence of the cyanogen bromide (CNBr) fragment CB I from the Ala chain of ricin D, the largest of three CNBr fragments, was established by manual Edman degradation of the peptides obtained by tryptic, chymotryptic or peptic digestion of fragment CB I. The total number of amino acid residues of fragment CB I accounted for 140 (54%) out of 260 residues in the Ala chain of ricin D.     

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.     

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).     

Synthesis of Penicillins and Cephalosporins by Penicillin Acylase Entrapped in Fibres

Tryptic peptides from two cyanogen bromide (CNBr) fragments CB II and CB III of the Ala chain of ricin D were sequenced by manual Edman degradation. Chymotryptic or peptic peptides from the two fragments were isolated by Dowex 1 x 2 column chromatography to obtain overlaps for the tryptic peptides, and the complete amino acid sequences of fragments CB II and III were established. The amino acid residues in fragments CB II and CB III accounted for 75 and 45 residues, respectively, of 260 residues in the Ala chain.

These sequences together with the sequence of fragment CBI described in the preceding paper established the complete sequence of the 260 amino acid residues in the Ala chain. Some structural characteristics of the protein are also discussed.     

Amino acid sequence of beta-galactosidase. X. Sequence of the COOH-terminal segment, CNBr peptides 18 to 24, residues 654 to 1021.

The sequence of the COOH-terminal third (omega) of beta-galactosidase is presented. The size of the 7 cyanogen bromide peptides of this segment is larger on the average, about 52 amino acid residues as compared to an average size of 42 for cyanogen bromide peptides in the whole molecule. Tyrosine, phenylalanine, and valine are low in this segment whereas alanine and lysine are high. This region has a slight excess of basic groups.     

Primary structure of streptococcal proteinase. I Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragments 1 to 4.

Tryptic and chymotroptic peptides were isolated and characterized from cyanogen bromide fragments 1 to 4 of streptococcal proteinase and subjected to sequence analysis by the Edman degradation, carboxy-peptidase digestion, and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. The results, together with the sequence data of the cyanogen bromide fragment 5 reported in the accompanying papers, provide the structural formula of streptococcal proteinase.     

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.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). II. The amino acid sequence and alignment of cyanogen bromide fragments IV, III, and I.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is the major protein constituent of the human plasma high density lipoproteins. Cleavage of this protein with cyanogen bromide yields four fragments, designated in the order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the first paper in this series, the amino acid sequence of the NH2-terminal fragment, CNBr II, is reported. In the present study, CNBr IV, III, and I, containing, respectively, 25, 36, and 94 amino acids were sequenced by conventional means. To establish the alignment of the cyanogen bromide fragments, apoLP-Gln-I was digested with trypsin and two of the three methionine-containing tryptic peptides were isolated. The amino acid sequence of apoLP-Gln-I is as follows: (see article). With the complete amino acid sequence available, a CPK space-filling model of apoLP-Gln-I was constructed. The protein was placed into an alpha helical conformation wherever the primary structure permitted. Thirteen helical regions were identified. These regions account for 70% of the amino acid residues of the protein. Each helix is characterized as having two faces (amphipathic). One is a polar face that occupies approximately 180 degrees of the surface of the helix and the other is a hydrophobic face that occupies the other 180 degrees of the helical surface. Similar amphipathic helices have been identified previously in the other lipoprotein-proteins that have known sequences. It is suggested that the amphipathic helical regions of apoLP-Gln-I are important in the binding of phospholipids in high density lipoproteins.     

Amino-acid sequence of the short subfragment-2 in adult chicken skeletal muscle myosin

The amino-acid sequence of a short subfragment-2 in the amino-terminal portion of subfragment-2 (S-2) derived from adult chicken skeletal muscle myosin was completely determined. Peptides cleaved by cyanogen bromide and by lysyl endopeptidase of S-carboxymethylated S-2, and hydrolytic peptides obtained with trypsin or dilute acetic acid of larger CNBr fragments were isolated and sequenced. This region was composed of 257 amino-acid residues, and hydrophobic and charged residue repeat units were found highly conserved and with a periodicity in 7 or 28 residues. This sequence of the short S-2 fragment of chicken skeletal muscle myosin was compared with the sequence of chicken and rat embryonic skeletal muscle myosins, rabbit skeletal and rabbit cardiac muscle myosin (alpha-myosin heavy chain), and 95.3%, 86.8%, 89.9% and 94.2% sequence identities were observed, respectively.     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.     

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.