The primary structure of actin from rabbit skeletal muscle. Five cyanogen bromide peptides, including the NH2 and COOH termini.

As a part of a study of the complete amino acid sequence of actin we have determined the sequences of five cyanogen bromide peptides, which together contain 158 amino acid residues, including the two ends of the molecule. The five peptides are: CB-13 (residues 1 to 44 in the intact chain), CB-11 (residues 83 to 119), CB-12 (residues 228 to 268), CB-8 (residues 283 to 298), and CB-9 (residues 355 to 374). Each of the peptides except CB-11 has one sulfhydryl group, and these peptides thus account for 4 of the 5 cysteines in actin. The reactivity of actin --SH groups toward N-ethylmaleimide was investigated, and it was found that Cys-373 (in CB-9 adjacent to the COOH-terminal phenylalanine) is the first to react with this reagent.     

Structural studies on rabbit skeletal muscle actin. Ordering of the peptides produced by cleavage with cyanogen bromide.

The 17 peptides produced by cleavage of actin with cyanogen bromide have been ordered with regard to their sequence in the actin molecule. Tryptic digestion of actin followed by isolation of the methionine-containing "overlap" peptides permitted the unique alignment of most, but not all of the cyanogen bromide peptides. However, maleylation of the actin molecule followed by tryptic digestion and isolation of methionine-containing peptides from maleylated actin permitted the proper placement of the remaining cyanogen bromide peptides. The ordering of cyanogen bromide peptides, together with the amino acid sequence of the individual peptides, constitutes the entire amino acid sequence of rabbit skeletal muscle actin (ELZINGA, M., COLLINS, J. H., KUEHL, W. M., and ADENLSTEIN, R. S. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,2687-2691).     

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.     

The primary structure of actin from rabbit skeletal muscle. Three cyanogen bromide peptides that are insoluble at neutral pH.

Three of the 17 peptides produced when actin is treated with cyanogen bromide are sparingly soluble at pH values near neutrality. They were separated from more soluble peptides at pH 6.0 on a column of Sephadex G-10. The soluble peptides were excluded from the gel and emerged at the void volume, while the insoluble peptides were "washed off" by the formic acid in which the sample was applied. The three insoluble peptides were sequenced as a group by studying peptides generated by tryptic and chymotryptic digestion of the mixture, and peptic digestion of the partially resolved peptides. The three peptides are: CB-15 (residues 133 to 176), CB-16 (residues 325 to 354), and CB-17 (residues 191 to 227).     

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.     

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.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.     

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.     

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.     

Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase

The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.     

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---.     

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.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment

The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.     

Primary structure of the elongation factor G from Escherichia coli. IX. Structure of peptides generated by cyanogen bromide cleavage of the G-factor isolated on thiol-activated sepharose and of the products of the G-factor cleavage at Asp-Pro bonds. Complete primary structure

The amino acid sequence of cysteine- and cystine-containing peptides resulting from cleavage of the G-factor by cyanogen bromide has been determined. For structure analysis cyanogen bromide peptides were further degradated using trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease, or limited acid hydrolysis. The products of the G-factor cleavage at Asp-Pro bonds were also studied. The obtained data together with those published earlier permitted to establish the complete primary structure of the elongation factor G. The polypeptide chain consists of 701 amino acid residues and has molecular mass of 77321,46.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).     

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.     

Cytochrome c from Schizosaccharomyces pombe. 2. Amino-acid sequence.

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end. After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.     

Structural characterization of the calcium binding protein s100 from adipose tissue

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.     

Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.