Primary structure of otter (Lutra lutra L.) myoglobin. 1. Soluble peptides of a tryptic hydrolysate

The amino acid sequence of 24 soluble tryptic peptides has been determined for the main chromatographic component of otter myoglobin. Fractionation, identification and peptide isolation have been made by fingerprints. The isolated peptides contain in total 111 amino acid residues.     

Amino acid sequence of Ondatra myoglobin (Ondatra zibethica). Characteristic features of myoglobins from semiaquatic animals

Based on the amino acid composition of globin, amino acid analysis and N-terminal sequencing of peptides as well as a comparative analysis of the primary structure of beaver, coypu rat and otter myoglobins with the use of the fingerprinting technique, the amino acid sequence of the major component of ondatra myoglobin including 153 amino acid residues was reconstructed. The results of a comparative analysis of the primary structure of myoglobin and the peculiarities of the functional morphology of myoglobins from semi-aquatic animals and sperm whale and the role of amino acid substitutions in the spatial structure of ondatra myoglobin are discussed.     

The myoglobin of primates. VIII. Gorilla gorilla beringei (eastern highland gorilla).

On aligning the tryptic peptides of the myoglobin from a gorilla with the homologous human peptides, one amino acid difference was found. By dansyl-Edman degradation this was shown to be at position 22, i.e. at a position other than those where man, chimpanzee and gibbon differ from one another.     

The primary sequence of chicken myoglobin (Gallus gallus).

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.     

The amino acid sequence of myoglobin from skeletal muscles of red deer(Cervus elaphus)

Red deer myoglobin has been fragmented by restricted tryptic digestion and by treatment with cyanogen bromide. The fragments have been separated by gel permeation. The core peptide derived from cyanogen bromide cleavage have been further digested with trypsin and the resulting peptides have been separated on Dowex 1X2. All fragments have been characterized by their amino acid composition, by determination of their N-terminal sequence using automatic Edman degradation and of their C-terminal sequence following the kinetics of amino acid cleavage by carboxypeptidases A and B. The complete sequence has been found to be identical with the already known sequence of sheep myoglobin except for residue 145 which is Gln in red deer globin and Glu in sheep globin. Reinvestigation of the corresponding sequence in sheep globin has shown that residue 145 of sheep globin is also Gln.     

Sequences of the soluble tryptic peptides from myoglobin of yellowfin tuna (Thunnus albacares).

1. Amino acid sequences of the soluble tryptic peptides of yellowfin tuna myoglobin, comprising 60% of the total residues, are presented. 2. The amino terminus is acetylated as shown by Fourier transform nuclear magnetic resonance spectroscopy of an N-terminal dipeptide. 3. Comparison of peptide sequences from yellowfin tuna myoglobin with corresponding regions of mammalian myoglobins shows obvious homology around the heme-attachment site and the carboxyl terminus, but marked dissimilarity is evident at other locations, such as the amino terminal region.     

Complete amino acid sequence of the major component myoglobin of finback whale (Balaenoptera physalus)

The complete amino acid sequence of the major component myoglobin from finback whale, Balaenoptera physalus, was determined by the automated Edman degradation of several large peptides obtained by specific cleavages of the protein. Three easily separable peptides were obtained by cleaving with cyanogen bromide at the two methionine residues and one large peptide was isolated after cleavage with (2-p-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. More than 60% of the covalent structure was established by the sequential degradation of three of these peptides and the apomyoglobin. An additional 30% of the primary sequence was established with peptides obtained from tryptic digestion of both the apomyoglobin and the acetimidoapomyoglobin, and the final 10% of the sequence was completed after digestion of the two larger cyanogen bromide peptides with S. aureus strain V8 protease. This myoglobin differs from that of the sperm whale, Physeter catodon, at 15 positions, from that of the arctic minke whale, Balaenoptera acutorostrata, at 3 positions, and from that of the California gray whale, Eschrichtius gibbosus, at 4 positions. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

Reactions of the protein radical in peroxide-treated myoglobin. Formation of a heme-protein cross-link

Reaction of horse myoglobin with H2O2 oxidizes the iron to the ferryl (Fe(IV) = O) state and produces a protein radical that is rapidly dissipated by poorly understood mechanisms. As reported here, the reaction with H2O2 results in covalent binding of up to 18% of the prosthetic heme group to the protein. The chromophore of the protein-bound prosthetic group is very similar to that of heme itself. High performance liquid chromatography of tryptic digests indicates that the formation of heme-bound peptides is associated with disappearance of the peptide with the sequence YLE-FISDAIIHVLHSK corresponding to residues 103-118 of horse myoglobin. Amino acid analysis, terminal amino acid sequencing, and liquid secondary ion mass spectrometry establish that the heme is primarily attached to this peptide. The heme appears to be bound to the tyrosine residue because the tyrosine is the only amino acid that disappears from the amino acid analysis. The mass spectrometric data indicates that the heme-peptide is formed without addition or loss of an oxygen or other major structural fragment. The site of attachment to the heme group has not been unambiguously determined, but the heme vinyl groups are not essential for the reaction because equal cross-linking is observed in H2O2-treated mesoheme-reconstituted myoglobin. The results are most consistent with binding of tyrosine 103 to a meso-carbon of the prosthetic heme group.     

The myoglobin of the Cape hunting dog (Lycaon pictus).

Tryptic and other peptides from the myoglobin of the Cape hunting dog (Lycaon pictus) have been aligned with the sequence of the myoglobin of the domestic dog. One amino acid difference was found which was confirmed by dansyl-Edman degradation. The five carnivore myoglobins now known have been integrated into an evolutionary cladogram, trying to trace the pathways of mutations, and a possible ancestral myoglobin for the carnivores has been constructed.     

Amino acid sequence of a myoglobin from lace monitor lizard, Varanus varius, and its evolutionary implications

Myoglobin was purified from a muscle extract of lace monitor lizard, Varanus varius, by Sephadex G-75, followed by DEAE-cellulose column chromatography. The apomyoglobin was cleaved with cyanogen bromide. The largest fragment was further digested with pepsin, trypsin, and alpha-chymotrypsin. From the amino acid sequence of the cyanogen bromide fragments, together with those of tryptic peptides of apomyoglobin, the complete amino acid sequence of lizard myoglobin was deduced. To investigate the tetrapod and amniote origins, many possible phylogenetic trees were constructed using the myoglobin sequences, including those of map turtle and lace monitor lizard. The tree that requires the minimum number of nucleotide substitutions in their genes for the myoglobin sequences to have evolved from a common ancestor was different from the similarly most parsimonious trees for cytochrome c or for alpha-hemoglobin. The trees were different from each other and from the tree that best reflects current biological opinions.     

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.     

The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.     

The immunological activity of some of the chymotryptic peptides of sperm-whale myoglobin

1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin.     

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.     

Structure primaire de l'anhydrase carbonique érythrocytaire B humaine: II. Clivage par le bromure de cyanogène et séquence des résidus 1–148

The amino acid sequence of the IICNBr fragment of the human erythrocyte carbonic anhydrase B has been determined. This fragment contains the first 148 of the 260 residues of the N-acetylated single polypeptide chain of the protein. After tryptic hydrolysis of this fragment, eleven peptides have been isolated by gel filtration and chromatography on Dowex 50 W-X₂ or DEAE-Sephadex. Eight of them were identified with already sequenced peptides previously isolated from tryptic hydrolysate of the whole protein. The other three ones were obtained in pure form and sequenced. The combined amino acid content of these eleven peptides only account for 124 of the 148 amino acid residues in the IICNBr fragment. The tryptic attack of the maleylated IICNBr fragment gave three peptides as was expected from the number of arginine residues (2) in this fragment: two arginyl peptides (II₁, II₃) and one homoseryl peptide (II₂). They were purified by gel filtration. The unidentified 24 residue tryptic peptide has been isolated from the demaleylated II₂ tryptic hydrolysate and sequenced. The order of the twelve tryptic peptides of IICNBr fragment has been obtained by study of chymotrypsic peptides isolated from II₁ and IICNBr fragment.     

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity.

Clostripain catalyses the transpeptidation between benzoylarginin ethyl ester and amino acid amides, oligopeptides, insulin A- and B-chains and tryptic peptides of myoglobin at millimolar substrate concentrations. The reactions proceed with temporary accumulation of the products, followed by hydrolytic decomposition. The yield was not affected significantly by the type of N-terminal amino acid, but was diminished markedly by the negative charges of the amine components. The yields for natural peptides were linearly related to the charge density of the peptides.     

Crystal structure of myoglobin form a synthetic gene

Crystal have been grown of myoglobin produced in Escherichia coli from a synthetic gene, and the structure has been solved to 1.9 Å resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N-terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and roeientation of some surface side chains. Crystals of variant of the “synthetic” myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand and specificity.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. Study of the structure of peptides obtained by hydrolysis of fragment F1 with proteinase from Staphylococcus aureus

The fragment F1 resulting from the limited tryptic hydrolysis of the native molecule of cytochrome P-450 has been digested with Staphylococcus aureus protease. 24 peptides, covering the whole polypeptide chain of fragment F1, are isolated from the hydrolysate. Analysis of their amino acid sequence in combination with the earlier data on the structure of cytochrome P-450 chymotryptic peptides and fragment F1 tryptic peptides permitted to carry out a reconstruction of large peptide blocks of fragment F1 that comprise 252 amino acid residues.     

Amino acid sequence of beta-galactosidase. V. Isolation and sequences of chymotryptic peptides.

The amino acid sequence of 72 chymotryptic peptides isolated from 14C-, 3H-labeled carboxymethyl-beta-galactosidase has been determined. A variety of techniques were used in the isolation procedures including separation by solubility, size, and ion exchange and paper chromatography. These peptides contain approximatley 500 amino acids, range in size from 2 to 26 residues, and give overlaps with tryptic peptides of 16 to 55 residues. Peptides from this digest and those reported earlier from tryptic digests account together for the sequence of about 600 of the 1021 residues in the subunit.     

Primary structure of bovine erythrocyte carbonic anhydrase CI. I. Tryptic peptides]

Bovine erythrocyte carbonic anhydrase CI consists of 259 amino acid residues including 18 lysines and 9 arginines. Its primary structure has been first investigated by isolation and sequence determination of the tryptic units. Acidification of the tryptic hydrolysate leads to the precipitation of 40% of the peptidic material. All the acid soluble peptides were isolated from the supernatant by chromatography on Dowex 50 W-X2 and Dowex 1-X2 followed by purification of heterogeneous fractions. Two of the three acid insoluble peptides were obtained in a pure form from the whole tryptic hydrolysate by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex in alkaline medium. The sequence of the so isolated tryptic units has been determined with the exception of two of them obtained in a very poor yield.