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.     

Primary structure of rabbit skeletal muscle troponin-T. Sequence determination of the NH2-terminal fragment CB3 and the complete sequence of troponin-T.

The amino acid sequence of CB3, the NH2-terminal fragment of troponin-T, and the alignment of all six cyanogen bromide (CB) fragments are reported. Fragment CB3, comprised of 70 residues, has eight of the nine prolines of troponin-T. As observed in other proteins of the myofibrillar system, its NH2 terminus is blocked by an acetyl group. Methionine-containing "overlap" peptides isolated from a peptic digest of troponin-T as well as 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine cleavage of the protein were used to order the fragments as CB3-CB2-CB5-CB4-CB7-CB6. The complete sequence of troponin-T, a single polypeptide chain of 259 amino acids having a molecular weight of 30,500, is presented.     

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.     

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.     

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.     

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.     

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.     

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---.     

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.     

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.     

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.     

Isolation and Sequences of Peptic Peptides,and the Complete Sequence of Ile Chain of Ricin D

Sixteen peptic peptides, which contain arginine(s) or lysine, were isolated from cyanogen bromide fragments CB I and CB II of Ile-chain. Sequence determination has been performed on most of these peptides to provide overlaps for the tryptic peptides. Thus, complete amino acid sequence of Ile-chain consisting of 265 amino acid residues was determined. Some structural characteristics of the protein are also discussed.     

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.     

Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.     

Active center and subunit structure of transaldolase from Candida utilis.

Crystalline transaldolase (type III) isolated from Candida utilis is composed of two identical subunits, as shown by the following lines of evidence. 1. Tryptic digestion of the performic acid oxidized enzyme yields the number of ninhydrin- and arginine-positive peptides expected for identical subunits. 2. All attempts to separate both subunits by molecular weight or charge differences have failed. 3. Cyanogen bromide cleavage and sodium dodecyl sulfate gel electrophoresis of S-carboxymethylated transaldolase revealed four distinct peptides designated C₂ to C₅ according to their decreasing molecular weight and one additional peak, C₁, in low yield, presumably an aggregate or partially degraded peptide.By chromatography on Sephadex G-100 the maleylated cyanogen bromide digest from ¹⁴C-labeled β-giyceryl-transaldolase could be separated into four peptide peaks which have been analyzed for their amino acid composition. The largest peptide C₂ with a molecular weight of 16,800 was identified as the active site containing fragment. The four fragments together account for all amino acid residues in the entire protein.From transaldolase (type I) containing four methionine residues three cyanogen bromide peptides could be identified. By addition of the individual peptides a molecular weight of 37,100 ± 3500 could be calculated, which is half the molecular weight of the native enzyme. From experimental data presented so far both isoenzymes of transaldolase can be regarded as “half-of-the-sites” enzymes.     

The covalent structure of calf skin type III collagen. II. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB1,8,10,2(Positions 223--402).

The cyanogen bromide peptide alpha 1-(III)CB1,8,10,2 is 180 amino acid residues in length and occupies position 223 to 402 along the alpha 1(III) chain. In order to elucidate its amino acid sequence, alpha 1(III)CB1,8,10,2 was fragmented with hydroxylamine, protease from Staphylococcus aureus V8 and trypsin. Peptides necessary for sequence analysis with the automated Edman degradation were separated using molecular and ion exchange chromatography. Edman degradation of the hydroxylamine-derived fragments resulted in the elucidation of 80% of the entire sequence. The rest was completely established by sequence analysis of some protease V8 and trypsin-derived peptides.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.     

The complete amino acid sequence of a major wheat protein inhibitor of α-amylase

The complete amino acid sequence of one of the major wheat protein iso-inhibitors of α-amylase was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide and by digestion with trypsin, chymotrypsin, thermolysin and the Staphylococcus aureus V8 protease. The molecule consists of a single polypeptide chain of 123 residues. Both serine and alanine were found in position 65, and further minor examples of microheterogeneity were observed in four other residues.     

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.