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The amino acid sequence of myoglobin from cardiac muscle of the American alligator (Alligator mississippiensis) was established by alignment of overlapping peptides and dansyl-Edman degradation. The chain initiating methonine seems to be retained. Thus, this molecule has 154 amino acid residues rather than 153 as in other tetrapod myoglobins. Maximum parsimony analysis indicated that alligator myoglobin diverges more from both bird and mammal myoglobins than these latter do from each other. The phylogenetic implications are discussed with respect to a possible diphyletic origin for diapsid reptiles.

The amino acid sequences of skeletal muscle myoglobins from two old-world monkeys, Presbytis entellus and Erythrocebus patas, as well as one new-world monkey, Cebus apella wer inferred by homology of the tryptic and peptic peptides with the known sequence of human myoglobin and by selective dansyl-Edman degradation. These new sequences were examined phylogentically in conjunction with the 15 primate sequences already reported. It is clear that myoglobin evolution has been extremely conservative among cercopithecoid primates, so much so that the two surviving subfamilies cannot be distinguished using this protein.

Skeletal muscle myoglobin of the aardvark (Orycteropus afer) was isolated and its primary structure determined. The amino-acid sequence was then used in conjunction with previously established myoglobin sequences to evaluate the phylogenetic relationships of the aardvark. The most parsimonious trees constructed from this myoglobin sequence data either alone or when combined with lens alpha-crystallin A sequence data depict the aardvark lineage as one of the most ancient among Eutheria.

Myoglobin has been identified in the myocardium of the lamprey Petromyzon marinus, one of the most primitive of all vertebrates. This protein was isolated by ammonium sulphate fractionation and purified by successive chromatography on Ultrogel AcA 54, DEAE-Sephadex and CM-23 cellulose. The molecule differs substantially from the monomeric hemoglobins found in the lamprey as evidenced by its elution profile on DEAE-Sephadex and the fingerprint pattern of its enzymically-produced peptides. The functional significance of this protein in Agnatha is discussed.

The tryptic and peptic peptides from the myoglobin of the zebra (Equus burchelli) have been compared with those obtained from the myoglobin of the horse (Equus caballus). No differences in the myoglobin were found between these two species.

Myoglobin was purified from skeletal muscle of the pika (Ochotona princeps) and its primary structure was determined. This sequence was added to the set of 64 already known vertebrate myoglobin sequences and used to evaluate the phylogenetic position of the pika among other mammals, using a computerized search procedure based on maximum parsimony criteria. The pika is clearly related to the rabbit (Oryctolagus cuniculus), with which it is traditionally associated as a member of the order Lagomorpha. A monophyletic group composed of Lagomorpha, Scandentia, and Carnivora is a consistent feature of the dendrograms produced. An association of Carnivora and Lagomorpha casts doubt on the results of those investigations using rabbit antisera in systematic studies and depending on carnivores as an outside reference group for cladogram construction.

The substructure of intermediate filaments from bovine lens cortical fiber cells was investigated by electron microscopy. Native filaments and synthetic ones regenerated from the total cytoskeletal extract and from the three purified subunits were examined. The morphologies from these various sources were essentially identical, with the exception that filaments reconstituted from one of the purified polypeptides were much shorter, very contorted and showed strings of aggregated protein. The solid cylindrical, unbranching filaments consisted of a helical arrangement of at least two, 5 nm diameter strands. The evidence indicated that each strand was composed of two, 2 nm diameter protofilaments which were also helically constructed (right-handed) with a periodicity of 11.6 nm. Intermediate filament diameter varied widely (8-14.8 nm, average 11.3 nm) and in a direct, linear manner relative to the apparent progression (helical) angle of the strands across the filaments face. These conclusions were obtained from observations on negatively stained intact filaments and reconstituted 4.4 nm fibrils and on positively stained transverse sections of fixed and embedded filaments.

The complete amino acid sequence of skeletal myoglobin from the Asian elephant (Elephas maximus) is reported. The functional significance of variations seen when this sequence is compared with that of sperm whale myoglobin is explored in the light of the crystallographic model available for the latter molecule. The phylogenetic implications of the elephant myoglobin amino acid sequence are evaluated by using the maximum parsimony technique. A similar analysis is also presented which incorporates all of the proteins sequenced from the elephant. These results are discussed with respect to current views on proboscidean phylogeny.

The intermediate filaments from the bovine ocular lens can be fractionated into three 54000 dalton polypeptides (alpha, beta and gamma) which are present in equimolar quantities and differ from one another with respect to isoelectric point, two-dimensional peptide map pattern and total amino acid composition. The polypeptides, for which the name lentin is proposed, were purified from the 8 M urea extract of the water-insoluble residue of the cortical fiber cells by ion-exchange chromatography on DE- and CM-cellulose. The regeneration of native-appearance filaments from each purified fraction, or combinations thereof, definitively established the three polypeptides as the subunit species. Additionally, small diameter 'protofilaments' were regenerated when one or two fractions were utilized, but not when all three fractions were combined. This, together with the fact that the stoichiometric yield of the three species was invariably equimolar, suggests that alpha, beta, and gamma may be distinct entities not related by artifactual interconversion.

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.

The primary structure of the major component of human skeletal muscle troponin C has been established. The troponin C was purified by ammonium sulphate and isoelectric fractionation, followed by two chromatographic steps on DEAE Sephadex. The sequence was determined from the different overlapping enzymic peptides and by dansyl-Edman degradation. The only difference between rabbit skeletal muscle troponin C and the major component of human skeletal troponin C was found at position 112: Ala (rabbit), Pro (human). The partial amino acid sequence of the first 86 residues of the minor component of human skeletal troponin C was found to resemble the troponin C from bovine cardiac muscle. The only difference between them, has tentatively been located at position 62: Glu (human), Asp (bovine). These similarities suggest that troponin C is, from the point of view of molecular, one of the most conservative proteins so far studied.

The primary structure of the myoglobin of the prosimian Lorisidae Perodictius potto edwarsi (potto) was studied. Tryptic, chymotryptic, peptic, subtilisin and thermolysin peptides were aligned against the sequence of human myoglobin. Sixteen differences were found which were confirmed by sequential analysis. On comparison of the West African potto with two other prosimian myoglobins known so far, there were 12 differences between the potto and the galago (East African) and 18 differences between the potto and the sportive lemur (Madagascar).

Amino acid sequence determination of elephant myoglobin revealed the presence of the unusual substitution E7 His leads to Gln. Stereochemical analyses suggest that the most suitable residue which can functionally substitute for His at this position in vertebrate globins is Gln. Physiochemical studies imply that the slower rate of autooxidation of elephant myoglobin is the result of this substitution which may confer some selective advantage on the species. Comparative sequence data of paenungulate myoglobins suggest that the His leads to Gln mutation probably occurred in an ancestor of Elephantinae.

To the analysis of three prosimian myoglobins reported previously [1, 2] that of the slow loris (Nycticebus coucang) has been added. A cladogram has been constructed utilising the new information to arrive at a possible ancestral prosimian myoglobin. Within the primates only the four prosimians share residues of threonine at position 34 and of isoleucine at position 142. During tryptic digestion of the loris myoglobin, some hydrolysis was observed at bonds not usually considered to be susceptible to the action of this enzyme: 52 Pro-53 Asp, and 120 Pro-121 Gly.

The effects of distal glutamine (E7) replacement in elephant myoglobin were studied by comparing the temperature-dependent nitrosyl electron spin resonance spectra, redox potentials, and the acid-alkaline equilibria of elephant and human myoglobins. For myoglobins containing a distal histidine, the nitrosyl ESR spectra do not exhibit superhyperfine splitting until near liquid helium temperatures (Yoshimura, T., Ozaki, T., Shintani, Y., and Watanabe, H. (1979) Arch. Biochem. Biophys. 193, 301-313). Studies presented here show that the ESR spectra of nitrosyl elephant myoglobin exhibit 9-line superhyperfine splitting well above liquid nitrogen temperatures, similar to the temperature profiles of isolated heme complexes (Morse, R.H. (1980) Fed. Proc. 39, 2006). It is concluded that the shift in the spectral equilibrium to higher temperature indicates a diminished interaction between NO and the distal position in elephant myoglobin. In addition, the redox potential of elephant myoglobin was found to be nearly 100 mV greater than that of human myoglobin, and the pKa of the acid-alkaline equilibrium (oxidized myoglobin) was 8.5, being 0.4 unit less than that of other vertebrate myoglobins. These different reactivities between elephant and human myoglobins are discussed based on the nature of charge interactions between polar ligands and distal glutamine and histidine.

Alpha and beta tropomyosins were isolated from postmortem adult human psoas and pectoralis major muscles. 31P nuclear magnetic resonance and amino acid analysis were used to show that 10% of the major alpha tropomyosin component was phosphorylated. The 31P NMR spectra also suggested that human beta tropomyosin was phosphorylated, but to a lesser extent.

The thirteen year history of assessing evolution by amino acid sequence analysis has made apparent the limitations imposed upon this system by the finite nature of the characters. This finiteness exists on several levels and ultimately expresses itself as parallelism, back mutation and the retention of primitive characters in the sequences of proteins from present day species and the putative ancestral protein chains. Sequence analysis shares these problems with other molecular approaches, but because it is concerned both with the nucleotide substitutions in the genome and with the functional roles of proteins, it has unique advantages. For example, the large fluctuation in the rate of fixation of mutations in a protein's evolution can be detected and used to point out the unreliability of any molecular clock for estimating divergence dates. Moreover, when consideration is given to studies which assign functional significance to specific amino acid sites in a protein, changes in function during the descent of a protein can be appreciated and their significance correlated with organismal evolution.

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 myoglobin of a gibbon has been compared with that of man. There was only one difference, residue 23 (B4) which is Gly in man was found to be Ser in the gibbon. Two corrections have also been made to the previously published sequence of human myoglobin (A.E. Romero Herrera and H. Lehmann, Nature, New Biol., 232 (1971) 149).