The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). I. The amino acid sequence of cyanogen bromide fragment II.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is one of the major protein constituents of human plasma high density lipoproteins. The protein has 245 amino acid residues, including 3 residues of methionine, and is lacking isoleucine, cystine, and cysteine. Cleavage of apoLP-Gln-I with cyanogen bromide yields four fragments, designated in their order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the present study, we report the complete amino acid sequence of the NH2-terminal fragment, CNBr II, a peptide that contains 90 amino acid residues.     

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.     

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.     

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.     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

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 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.     

Amino acid sequence of beta-galactosidase. VIII. Sequence of the NH2-terminal segment, CNBr peptides 1 to 9, residues 1 to 377.

The amino acids in 9 cyanogen bromide peptides have been placed in sequence starting from the NH2 terminus. The peptides account for residues 1 to 377 of the whole protein and include the largest (CNBr7, 119 residues) and the smallest (CNBr1, 2 residues) of the cyanogen bromide peptides. This region contains only 3 of the 20 lysine residues in the polypeptide chain. A high proportion of charged groups are present (28 of 66 arginine, 28 of 60 glutamic acid, and 24 of 65 aspartic acid residues).     

Studies on cytochrome c oxidase, IX. The primary structure of polypeptide VIa

The complete amino acid sequence of the cytoplasmic polypeptide VIa of cytochrome c oxidase from beef heart is described. The primary structure of this component of complex IV of the respiratory chain is elucidated by isolation and sequencing of overlapping glutamic acid, arginine, tryptophan and methionine fragments obtained by cleavage with Staphylococcus aureus protease, protease from submaxillaris glands of mice, 2-iodosylbenzoic acid and cyanogen bromide. The chain length of polypeptide VIa is 98 amino acids, the resulting molecular mass of 10670 Da. The hydrophilic protein does not contain a hydrophobic membrane penetrating sequence domain. Its function in the respiratory complex IV is unknown.     

Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins.

The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.     

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.     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

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.     

Glyceraldehyde-3-phosphate dehydrogenase. Investigation on the regions responsible for self-assembly of subunits

1. In glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) the four S-loop form the core of the tetramer. 2. Amino acid sequence of the S-loop of the regions of GAPDH from carp muscle was established through the analysis of tryptic digests of the enzyme treated alternatively with bromocyanate and o-iodosobenzoic acid. 3. Enzyme had been oxidized with performic acid. After treatment with trypsin the peptide mixture was fractionated into fragments. 4. CNBr cleavage of this enzyme was performed after S-carboxymethylation. The respective cyanogen bromide fragments have been isolated and characterized. 5. The procedure of protein fragmentation by o-iodosobenzoic acid used to split tryptophanyl peptide bonds. 6. Each peptide obtained after enzymatic or chemical fragmentation was purified to homogeneity by Bio-Gel or Sephadex chromatography, high voltage electrophoresis and descending paper chromatography and characterized by electrochromatography, N- and C-terminal sequence and amino acid composition. 7. The results are compared with those obtained from studies on GAPDH from other sources.     

The covalent structure of calf skin type III collagen. VI. The amino acid sequence of the carboxyterminal cyanogen bromide peptide alpha 1(III)CB9B (position 928--1028).

The C-terminal cyanogen bromide peptide alpha 1(III)CB9B is 101 amino acid residues in length and occupies position 928--1028 along the alpha 1(III) chain. For sequence analysis, alpha 1(III)CB9B was fragmented with trypsin and chymotrypsin. The peptides obtained were separated using molecular sieve and ion exchange chromatography and sequenced using the automated Edman degradation procedure.     

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.     

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome).     

The covalent structure of hepatic microsomal epoxide hydrolase. I. Isolation and characterization of the cyanogen bromide fragments

Microsomal epoxide hydrolase was purified to homogeneity from phenobarbital-induced rabbit liver for the purpose of determining the complete amino acid sequence. All of the expected 11 cyanogen bromide fragments of epoxide hydrolase were isolated by a combination of gel filtration and high pressure liquid chromatography. Each was characterized by its amino acid composition and NH2-terminal amino acid sequence. The complete amino acid sequences of the eight small fragments, from 5-29 residues, were determined.     

The amino acid sequence of troponin I from rabbit skeletal muscle.

The complete amino acid sequence of rabbit skeletal muscle troponin I was determined by the isolation of the cyanogen bromide fragments and the tryptic methionine-containing peptides. Troponin I contains 179 amino acid residues and has a molecular weight of 20864. Its N-terminus is acetylated. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50055 (23 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.     

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.