Barry N. Jones

Evolution of the amino acid substitution in the mammalian myoglobin gene

SummaryMultivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.

Complete amino acid sequence of the myoglobin from the pacific spotted dolphin, Stenella attenuata graffmani

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Ultrapure cyanogen bromide-cleaved glucagon: Isolation in high yield by ion-exchange chromatography☆

Abstract Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.

Complete amino acid sequence of the myoglobin from the Pacific sei whale, Balaenoptera borealis.

The complete amino acid sequence of the major component myoglobin from Pacific sei whale, Balaenoptera borealis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apomyoglobin, over 75% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by the sequence analysis of peptides that resulted from further digestion of the amino-terminal and central cyanogen bromide fragments. The amino-terminal fragment was specifically cleaved at its two tryptophanyl residues with N-chlorosuccinimide and the central cyanogen bromide fragment was cleaved at its glutamyl residues with staphylococcal protease and at its single tyrosyl residue with N-bromosuccinimide. The primary structure of this myoglobin proved identical with that from the gray whale but differs from that of the finback whale at four positions, from that of the minke whale at three positions and from the myoglobin of the humpback whale at one position. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Complete amino acid sequence of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris.

The complete primary structure of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at its two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage with trypsin of the citraconylated apomyoglobin at its three arginine residues. Further digestion of the central cyanogen bromide peptide with S. aureus strain V8 protease and the 1,2-cyclohexanedione-treated central cyanogen bromide peptide with trypsin enabled the determination of the remainder of the covalent structure. This myoglobin differs from the cetacean myoglobins determined to date at 12 to 17 positions. These large sequence differences reflect the distant taxonomic relationships between the goose-beaked whale and the other species of Cetacea the myoglobin sequences of which have previously been determined.

Reassignment of residue 122 in the myoglobin from the killer whale,Orcinus orca

SummaryThe complete amino acid sequence of the major component myoglobin from killer whale,Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.