Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Phylogenetic variation in the primary structure of hemoglobins

The amino acid compositions of homologous tryptic peptides, as well as the amino acid sequences of complete peptide chains, are compared in hemoglobins of different species. Special attention is given to the analyses of mammalian and fish hemoglobins. The multiplicity of hemoglobins in one species and the differences between the hemoglobins of different species are considered. These differences are discussed in view of base exchanges, deletions, insertions, and gene duplications. Enormous differences are found between any two single hemoglobin chains. The number of amino acids varies from 127 to 156 residues. In comparing all the hemoglobins, only eight amino acids are found in identical positions (invariant residues). All hemoglobins analyzed to date follow the rule of “isopolar substitution” of Perutz, Kendrew, and Watson. The results are summarized and considered with regard to certain parallels in the morphology.     

Myoglobin of the shark Heterodontus portusjacksoni: isolation and amino acid sequence

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

A comparison of the reproductive cycle of three species of Lemur.

The duration of the luteal phase and the 'proestrus' phase in three species of lemurs (Lemur macaco, Lemur catta, and Lemur variegatus) was determined by serum progesterone measurement. Total immunoreactive estrogens were measured and remained below 0.20 ng/ml except for an apparent peak of short duration during estrus. Circulating testosterone levels in males confirm previously described seasonal gonadal function. The reproductive cycle of the female lemur is discussed and compared to the cycles of estrous and menstrual animals.     

Genetically engineered Mengo virus vaccination of multiple captive wildlife species

Encephalomyocarditis virus (EMCV), has caused the deaths of many species of animals in zoological parks and research institutions. The Audubon Park Zoo, (New Orleans, Louisiana, USA) attempted vaccination of several species with a killed EMCV vaccine with mixed results. This paper reports an attempt at vaccination against EMCV using a genetically engineered, live attenuated Mengo virus (vMC0) at the Audubon Park Zoo and Miami Metro Zoo, (Miami, Florida, USA) from December 1996 to June 1997. Several species of animals were vaccinated with vMC0, which is serologically indistinguishable from the field strain of EMCV. Serum samples were taken at the time of vaccination and again 21 days later, then submitted for serum neutralization titers against EMCV. The vaccinate species included red capped mangebey (Cercocebus torquatus), colobus (Colobus guereza), angolan colobus (Colobus angolensis), ruffed lemur (Lemur variegatus ruber and Lemur variegatus variegatus), back lemur (Lemur macaco), ring-tailed lemur (Lemur catta), siamang (Hylobates syndactylus), diana guenon (Cercopithicus diana), spider monkey (Ateles geoffroyi), common marmoset (Callithrix jacchus), talapoin monkey (Cercopithecus talapoin), Brazilian tapir (Tapirus terrestris), Baird's tapir (Tapirus bairdii), Malayan tapir (Tapirus indicus), dromedary camel (Camelus dromedarius), bactrian camel (Camelus bactrianus), gerenuk (Litocranius walleri), guanaco (Lama glama guanicoe), black duiker (Cephalophus niger), Vietnamese potbellied pig (Sus scrofa), babirusa (Babyrousa babyrussa), collard peccary (Tayass tajacu), and African crested porcupine (Hystrix africaeaustralis). The vaccine response was variable, with high virus neutralizing antibody titer responses in some primate species and mixed to poor responses for other species. No ill effects were seen with vaccination.     

Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains

The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.     

Homeothermic fish and hemoglobin: primary structure of the hemoglobin from bluefin tuna (Thunnus thynnus, Scromboidei)

Some fish are warm-bodied, e.g. the bluefin tuna (Thunnus thynnus), which has a muscle temperature 12-17 degrees C higher than its environment. This endothermy is achieved by aerobic metabolism and conserved by means of a heat-exchanger system. The hemoglobins of bluefin tuna are adapted to these conditions by their endothermic oxygenation, thus contributing to the preservation of the body energy. This is a new and so far unique property of tuna hemoglobin. The primary structure of the alpha and beta chains of bluefin tuna hemoglobins is presented. The sequence was determined after enzymatic and chemical cleavages of the chains and sequencing of the peptides in gas- and liquid-phase sequencers. The alpha chains consists of 143 residues and are N-terminally acetylated. The beta chains have 146 amino acids and show two ambiguities at positions 140 and 142. The alpha chains differ from the human alpha chains in 65 amino-acid residues, the beta chains in 76. The hemoglobins of bluefin tuna, carp and man are compared and their different physiological properties are discussed in relation to the sequence data. From the primary structure of tuna hemoglobins, it is possible to propose a molecular basis for their peculiar endothermic transition from the T to the R structure.     

Hemoglobins, XXIX. Sequence analysis of a dimeric hemoglobin (erythrocruorin), CTT-X, of Chironomus thummi thummi (Diptera).

The amino acid sequences of one of the dimeric hemoglobin components, CTT-X, of Chironomus thummi thummi (Diptera) are given. The sequences were determined by automatic Edman degradation of tryptic peptides and peptides obtained by specific chemical cleavages. CTT-X has two different polypeptide chains, each with 151 amino acid residues. The two polypeptide chains differ only in one amino acid. The sequences are discussed in the light of the sequences of other related heme-proteins.     

Prosimian hemoglobins: IV. The structural difference responsible for the hemoglobin phenotype of Lemur catta

The ring-tailed lemur, Lemur catta, shows a two-component hemoglobin phenotype after alkaline electrophoresis. A difference in the amino acid sequence of the isolated α-globins was observed at position 15 (α I-Gly, α II-Lys) and can account for the electrophoretic pattern of two hemoglobin components. Only one other amino acid difference was found in the sequence of the two globin chains: a neutral substitution occurs at position 53 (α I-Gly, α II-Ala). The complete primary structures of the duplicated α-globin chains of Lemur catta are presented.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 2. Amino acid sequence of the alpha chain of Hb1

The complete amino acid sequence of the alpha chain of the main hemoglobin of the Antarctic fish Notothenia coriiceps neglecta (family Nototheniidae) has been determined. It consists of 142 residues; an acetylated seryl residue is at the amino terminal. The molecular mass is 15,519 Da. In comparison with alpha-chain sequences of non-Antarctic poikilothermic fish hemoglobins, the homology appears to be significantly lower than that existing among the latter species. A higher homology has been found with the alpha-chain sequence of the non-poikilothermic bluefin tuna.     

Carnivora: the primary structure of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae)

The primary structure of the alpha- and beta-chains of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae) is described. The chains were separated directly from hemoglobin by RP-HPLC. After tryptic digestion of the chains, the peptides were separated by RP-HPLC. Amino acid sequences were determined by Edman degradation in liquid and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. Paguma and human hemoglobin differ with respect to 23 amino-acid residues. Some of these amino-acid substitutions, which occur in both the alpha- and beta-chains, occur at contact sites between the subunits, and at the binding sites of heme and of organic phosphate, as well as involving residues responsible for the alkaline Bohr effect.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.     

Prosimian hemoglobins--V. The primary structures of the alpha-I, alpha-II and beta-hemoglobin chains of Hapalemur griseus, with a note on the classification of Microcebus

1. The duplicated adult hemoglobins were isolated from a mature Hapalemur griseus and the constituent chains prepared. Sequence analysis of the isolated alpha-globins showed that the alpha-I and alpha-II chains differed by a glycine for lysine substitution at position 15. 2. The complete amino acid sequence of the single adult beta-globin of Hapalemur griseus was determined. The beta-globin sequence of Hapalemur griseus clusters with those of other authentic lemurs, and is clearly separated from the sequences characteristic of lorisiform primates. 3. Partial sequence analysis of the beta-globin of Microcebus murinus showed only a single amino acid difference when compared to the Hapalemur globin. 4. Partial sequence analysis of the alpha-globin of Microcebus murinus showed only three amino acid residues that are not found in other lemuriform alpha-globins; two of these are unique to Microcebus among all known prosimian alpha-globin sequences. 5. The Microcebus alpha- and beta-globins are more similar to the homologous lemuriform sequences than they are to lorisiform sequences.     

Deoxygenation-linked association of a tetrameric component of chicken hemoglobin.

Deoxygenation-dependent association of hemoglobin tetramers appears to be widespread among amphibians, reptiles, and possibly all or most birds. The evidence for this conclusion depends largely on oxygen equilibria of whole blood which have Hill coefficients that reach values as high as 5-7 at 80-90% oxygenation. Computer simulation of the sedimentation velocity behavior of the major components A and D of chicken hemoglobin shows that component D but not A self-associates to form dimers of tetramers. The gradient profiles at pH 7.5 were satisfactorily fitted with an association constant of 1.26 x 10(4) M-1 and sedimentation coefficients of 4.63 and 7.35 S for tetramer and (tetramer)2, respectively. Since components A and D share common beta chains we conclude that tetramer-tetramer contacts must depend on surface residues of the alpha chains. Comparison of the amino acid sequences of the alpha D and alpha A chains of the hemoglobins from 12 avian species ranging from sparrow to ostrich shows that 20 residues are conserved in the alpha D chains but not in the alpha A chains. Nine of these (45%) are clustered between positions E20 and FG2. Four of the latter, Lys71 (E20), Asn75 (EF4), Gln78 (EF7), and Glu82 (F3) are conserved in all alpha D chains even though they do not appear to participate in intratetramer contacts. Molecular modeling indicates that residues Lys71, Gln78, and Glu82 of the alpha chain are strong candidates for the primary tetramer-tetramer contacts.     

Photosystem II of rye. Nucleotide sequence of psbB and psbH genes, coding 47-kDa of chlorophyll(a)-binding and 10-kDa phosphorylated subunits

Chloroplast DNA was isolated from rye seedlings by the non-aqueous method. The region of rye ctDNA which comprises two genes psbB and psbH encoding polypeptide subunits of photosystem II (47 kappa l) Chl alpha -binding protein (CP alpha -1) and 10 kD phosphoprotein, respectively) and two ORFs in the opposite strands in the psbB--psbH spacer region encoding hydrophobic peptides with strongly charged C-terminal segments was sequenced. The deduced amino acid sequences of polypeptide products of the genes were compared with those of different plant species (in case of the psbB product also with sequence of a cyano-bacterium Synechocystis) and revealed some highly conservative amino acid residues and regions of polypeptide chains, which apparently play essential role in the interaction with other PS II subunits and in the binding of chlorophyll molecules. Some speculations are made on the possible function of the peptides encoded by the two ORFs.     

Studies on monotreme proteins. VI. Amino acid sequence of the beta-chain of haemoglobin from the platypus, Ornithorhynchus anatinus.

The amino acid sequence of the 146 residues of the beta-chain of the major haemoglobin from the platypus has been determined. The soluble peptides derived from the chain by tryptic digestion were isolated by paper ionophoresis and chromatography. The amino acid sequences were determined by the dansyl-Edman procedure or by further digestion with other enzymes. The tryptic peptides were aligned by homology with other beta-globins. There were 14 changes in sequence compared with echidna beta-chain. The number of changes in sequence compared with human beta-chain is 34 which is less than the 39 changes between human and platypus alpha-chains. Generally there are more changes between beta-chains; there are only three other examples reported where there are more changes between alpha-chains than beta-chains, these are of echidna, rabbit and dog globins. By comparison with the 'contact sites' in horse haemoglobin there is one change in beta-haem contacts, three changes in beta1-alpha1 contacts and no changes in beta2-alpha1 contacts. The date of divergence of the monotremes from the other mammals was estimated at 132 +/- 33 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha-chain sequences and the significance of this discrepancy to the validity of the method is discussed.     

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).     

Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity.

Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.