Isolation and characterization of fin whale prolactin.

A highly purified prolactin preparation has been obtained from fin whale pituitaries by extraction with acid acetone. salt precipitation, isoelectric fractionation, and exclusion chromatography on Sephadex G-100 and ion-exchange chromatography on DEAE-CELLULOSE. Fin whale prolactin was isolated in a yield of 250 mg/kg wet weight tissue. It was found to have a molecular weight (SDS disc gel electrophoresis) of 23,600 daltons and an alpha-helix content (circular dichroism) of 50%. The amino acid composition and circular dichroism spectra were very similar to those of porcine prolactin. The partial amino acid sequence has been determined by the method of fluorescein-isothiocyanate. Fin whale prolactin was found to be 80% as potent as ovine prolactin with regard to pigeon crop-sac assay.     

Primary structure of equine pituitary prolactin

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.     

Primary structure of chicken pituitary prolactin deduced from the cDNA sequence. Conserved and specific amino acid residues in the domains of the prolactins

The perform of chicken prolactin (PRL) deduced from the cDNA sequence contains a signal peptide of 30 amino acid residues followed by a mature PRL of 199 residues. Chicken PRL shows 77, 68, 67, 58, and 31% identity of amino acid sequence with whale, human, ovine, rat, and salmon PRLs, respectively. Elucidation of the primary structure of avian PRL enabled extended analysis of the specific and conserved amino acid residues and domains of the PRL molecules. The mammalian, teleostean, and avian PRLs share 32 common residues, and these conserved residues are observed to cluster in four distinct domains (PD1 to PD4), corresponding to four of five conserved domains of the growth hormones. Of the 32 residues, 8 residues in the PD2 and PD4 domains, including 4 cysteines, are conserved by other members of the growth hormone family, which indicates that these 8 residues may be essential for common structural features of the gene family. On the other hand, 13 other residues distributed among all four domains are conserved almost exclusively in the PRLs, suggesting that these residues are indispensable for specific binding of the PRLs to their receptors.     

cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins

We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

Primary structure of chum salmon prolactins: occurrence of highly conserved regions

The complete amino acid sequence of prolactin from the pituitaries of salmon (Oncorhynchus keta) has been determined. Salmon prolactin comprised two variants, I and II, which were separated by reverse-phase high-performance liquid chromatography. Each variant was reduced, S-carboxymethylated, and then cleaved with cyanogen bromide and enzymes. The resulting fragments were separated by reverse-phase high-performance liquid chromatography, as well as gel filtration, and subjected to sequence analysis by the dansyl-Edman method. Both variants contain 187 amino acid residues with two disulfide linkages at residues 46-160 and 177-187, lack a linkage in the N-terminal portion of mammalian prolactins, and differ from each other by the replacement of only four amino acid residues. Salmon prolactin (sPRL) shows 31% sequence identify with ovine prolactin. Moreover, four restricted regions, i.e., sPRL (3-21), (46-60), (68-83), and (160-178), encompass this significant conservatism between the teleost and the mammalian hormone, with identities of 47, 87, 62, and 68%, respectively. Such considerable identity between these distant phylogenic species strongly suggests that these regions may be responsible for the biological activity of prolactin.     

Purification and properties of whale thyroid-stimulating hormone III. Properties of isolated multiple components.

Properties of the four purified components of whale thyroid-stimulating hormone (TSH) have been compared. The amino acid composition shows close similarity among these components. Their hexosamine and sialic acid contents are of the same magnitude, whereas the neutral sugar composition differs somewhat from each other. The molecular weight of whale TSH determined by sedimentation equilibrium is 29,000, and no difference in molecular weight as well as in Stokes radius as determined by gel filtration has been detected among these four components. The amino acid and carbohydrate compositions of whale TSH resemble those of TSH from other species, especially those of non-primate mammalian TSH. Whale TSH contains, unlike bovine TSH but like human TSH, 1-2 residues of sialic acid as a constituent carbohydrate.     

Studies on pituitary lactogenic hormone. The primary structure of the porcine hormone.

The complete amino acid sequence of porcine lactogenic hormone has been elucidated. It consists of 199 amino acid residues with three disulfide bridges formed by residues 4-11, 58-174, and 191-199. The two tryptophan residues are in positions 91 and 150. A high degree of homology exists between the known structure of ovine prolactin and the porcine lactogenic hormone. This led to a re-examination of residues 82-90 in the ovine prolactin structure, where an extra leucine was found in position 88.     

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 complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

The amino acid sequence of the calmodulin obtained from sea anemone (Metridium senile) muscle

The amino acid sequence of the calmodulin obtained from sea anemone muscle was determined. The calmodulin was composed of 148 amino acid residues and its amino terminal was blocked. When compared with bovine brain calmodulin, the number of amino acid residues per molecule was the same and there were 3 replacements at residues 99 (Tyr → Phe), 143 (Gln → Lys) and 147 (Ala → Ser).     

The complete amino acid sequence of growth hormone of the bullfrog (Rana catesbeiana).

The primary structure of growth hormone (GH) isolated from the adenohypophysis of the bullfrogs (Rana catesbeiana) was determined. The hormone was reduced, carboxymethylated and subsequently cleaved with cyanogen bromide. Intact bullfrog GH was also digested with lysyl endopeptidase and trypsin. The resulting fragments were separated by reverse-phase high-performance liquid chromatography and subjected to sequence analysis using an automated gas-liquid sequencer employing the Edman method. Bullfrog GH was found to consist of 190 amino acid residues. The amino acid sequence determined is in accord with that deduced from bullfrog GH cDNA by Pan and Chang (1988) except for nine residues at positions 43-48, 73, 80 and 87. Sequence comparisons revealed that bullfrog GH is more similar to tetrapod GHs (e.g., 69% homology with sea turtle GH, 66% with chicken GH and 61% with ovine GH) than to GHs of teleosts (e.g., 35% homology with chum salmon GH and 33% with bonito GH) except for eel (52% identity). Bullfrog GH and prolactin exhibit a sequence homology of 25%.     

Calitoxin, a neurotoxic peptide from the sea anemone Calliactis parasitica: amino acid sequence and electrophysiological properties

We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.     

Isolation and certain properties of human pituitary prolactin]

A simple method of isolation of highly purified prolactin from acetonated preparations of anterior hypophysial lobes is described. The new method permits to obtain higher (about 10-fold) yields of the hormone, as compared to those obtained using previously described methods. Prolactin was extracted by acid aqueous acetone and was subsequently purified of extract by fractionation with acetone and NaCl and by isoelectric precipitation. The final stage of the hormone purification involved gel-filtration through Sephadex G-200; prolactin yield was 400 microgram per 1 hypophysis. The lactogenic activity of the hormone is 14 MU/mg; the sequence of N-terminal amino acid residues of prolactin is as follows: NH2-Leu-Pro-Ile-x-Pro-Leu(?)-Gly-Ala-.     

Expression of new members of the prolactin growth hormone gene family in bovine placenta. Isolation and characterization of two prolactin-like cDNA clones

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone. The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively. bPLP-I, bPLP-II, placental lactogens, prolactins (PRLs), and other prolactin-like proteins isolated from cow, mouse, and rat share 7 common amino acid residues. Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.     

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.     

A comparison of the properties of renin isolated from pig and rat kidney.

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.