Purification of bovine beta 2-microglobulin from colostrum and its complete amino acid sequence.

Bovine beta 2-microglobulin (beta 2-m) was purified from colostrum milk in two chromatographic steps: anion-exchange chromatography, and gel filtration. The amino acid sequence was determined to confirm that the purified protein was beta 2-m. A molecular weight of 11.8 kDa beta 2-m was estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bovine beta 2-m consists of 98 amino acid residues and contains one disulfide linkage connecting residues 25 and 79. The amino acid sequence determined by this study is different from a previously published sequence at three sites, but agrees with the amino acid sequence from cDNA. Thus, we can conclude that the amino acid sequence determined in this study is correct.

Somatolactin, a novel pituitary protein: isolation and characterization from Sparus aurata.

The complete amino acid sequence of somatolactin, a new pituitary protein belonging to the growth hormone/prolactin family, from the gilthead sea bream Sparus aurata has been determined. Somatolactin was isolated from the pituitary by alkaline extraction, gel filtration on a Sephadex G-100 column, and reversed-phase high-performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T column. The purified protein was confirmed to be somatolactin by immunoblotting using chum salmon somatolactin antisera. It was found that Sparus aurata somatolactin consists of two forms; one form (28 kD) is probably a glycosylated form, while the other (25 kD) is a simple protein form, as was found also in Atlantic cod. The somatolactin consists of 207 amino acids that show remarkable conservation among fish somatolactins.

Complete amino acid sequence of crocodile growth hormone.

This paper describes the isolation and complete amino acid sequence of growth hormone (GH) from the pituitary gland of the crocodile, Crocodylus novaeguineae. GH was detected in an alkaline extract of the pituitary by specific immunoblot reactivity with rabbit antisera against cattle and salmon GH and isolated in pure form by DEAE-cellulose chromatography and by reversed-phase high-performance liquid chromatography. The yield of the GH was 2.2 mg from 3.5 g of wet tissue. Crocodile GH consists of 190 residues and is highly similar to chicken, duck, and sea turtle GHs with 90-92% structural similarity, although it shows only 27% identity with crocodile prolactin. A molecular phylogenetic tree of GH from 39 vertebrate species is proposed on the basis of sequence comparison.

The complete amino acid sequence of pituitary cystatin from chum salmon.

Cystatin, a cysteine proteinase inhibitor, was isolated from chum salmon (Oncorhynchus keta) pituitary glands by ion-exchange chromatography on Mono-Q, gel filtration on Superdex 75, and reverse-phase HPLC on an ODS following ethanol-ammonium acetate extraction. Salmon pituitary cystatin was equipotent to chicken egg-white cystatin in the papain inhibitory assay. The cystatin consists of 111 amino acid residues with two disulfide linkages formed between 66-75 and 89-109, and has 43% identical sequences with chicken egg-white cystatin with consensus sequences of reactive sites, Gly(4), Gln-X-Val-X-Gly (48-52), and Ile(Val)-Pro-Trp (96-98).

The primary structure of sturgeon prolactin: phylogenetic implication.

The complete amino acid sequence of prolactin (PRL) from a chondrostean species, the sturgeon (Acipenser gueldenstaedti), has been determined. Sturgeon PRL was isolated from the pituitary glands by gel filtration on a Sephadex G-25 column and high-performance liquid chromatography on a reverse-phase column following acid-acetone extraction. Sturgeon PRL was identified by immunoblot reactivity using antisera against salmon and ovine PRL. It consists of 204 amino acid residues, which is the largest among known PRLs, and contains three disulfide bonds corresponding to those of tetrapod PRLs. Sequence comparison with PRLs from other vertebrates revealed that sturgeon PRL has slightly higher sequence identities (35-46%) with teleost PRLs than with tetrapod PRLs (30-40%). These structural characteristics imply that an ancestor of the ray-finned fishes had PRL with three disulfide bonds and at some point after divergence of Chondrostei, the disulfide bond in the amino-terminus of PRL was lost.

Lungfish prolactin exhibits close tetrapod relationships.

This paper describes the isolation and the complete amino-acid sequence of prolactin (PRL) from the pituitary glands of African lungfish, Protoputerus aethiopicus. We purified the hormone from an alkaline extract of the pituitaries using a two-step chromatographic procedure by detecting specific immunoblot reactivity with rabbit antisera against salmon PRL. The lungfish PRL consists of 200 amino-acid residues. Sequence comparison revealed that the PRL shows 66% identities with amphibian, reptilian and bird PRLs, 57% with mammalian PRLs, and 38% with teleost (modern bony fish) PRLs. Moreover, the PRL contains three disulfide bonds homologous to those of tetrapod PRLs and differs from teleost PRLs which lack the amino-terminal disulfide bond. Thus, the structural features of lungfish PRL indicate a closer relationship to tetrapod PRLs than to teleost PRLs. All PRLs sequenced to date share 22 common amino acids, which may be important for the activities common to all PRLs.

Isolation of prolactin and growth hormone from the pituitary of the holostean fish Amia calva.

Pituitaries from adult male and female Amia calva (Order Holostei) were acid extracted and fractionated by gel filtration column chromatography and reversed-phase high performance liquid chromatography. This two-step isolation procedure yielded homogeneous pools of Amia prolaction (PRL) and growth hormone (GH). The amino acid composition of both purified polypeptides was determined. Primary sequence analysis of the first 22 positions at the N-terminal of Amia PRL revealed that this region has 63% sequence identity with eel PRL-1. The N-terminal region of Amia PRL lacks the disulfide bridge which is characteristic of tetrapod PRLs. Primary sequence analysis of the first 24 positions at the N-terminal of Amia GH revealed that this region has 62% sequence identity with eel GH and 54% sequence identity with both blue shark GH and sea turtle GH. Based on N-terminal analysis, it appears that Amia PRL and GH are more closely related to teleost PRLs and GHs than they are to tetrapod PRLs and GHs.

Flounder growth hormone: isolation, characterization, and effects on growth of juvenile rainbow trout.

A highly purified growth hormone (GH) was isolated from the pituitary of the Japanese flounder (Paralichthys olivaceus) following extraction with alkaline solution, gel filtration on Sephadex G-75, and high-performance liquid chromatography (HPLC) on a reverse-phase column. Flounder GH (flGH) has a molecular weight of 20 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and an isoelectric point of 7.1 by gel electrofocusing. The complete amino acid sequence was determined by Edman degradation of the peptide fragments generated by enzymatic digestion and chemical cleavage. The flGH comprises 173 amino acid residues with pyroglutamate at the N-terminus. Compared with other fish GHs, flGH has an uninterrupted deletion of 14 amino acid residues near the C-terminus. flGH is the smallest of growth hormones so far reported and is a potent stimulator of growth in juvenile rainbow trout.

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

Chemical identification of catfish growth hormone and prolactin.

Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.

Eel insulin: isolation, characterization and stimulatory actions on [35S]sulphate and [3H]thymidine uptake in the branchial cartilage of the eel in vitro.

Our previous studies have shown that mammalian and salmon insulins stimulate sulphate uptake by cultured eel cartilage, suggesting the possible involvement of insulin in the regulation of cartilage matrix synthesis. In the present study, homologous eel insulin was isolated and characterized, and its effects on cartilage matrix synthesis and DNA synthesis were examined in vitro. Insulin was extracted from eel pancreas with acid-ethanol, and subsequently purified by isoelectric precipitation at pH 5.3, gel filtration on Sephadex G-50, and reversed-phase high-performance liquid chromatography. The amino acid composition and complete sequence (50 residues) of eel insulin revealed high homology to teleostean and mammalian insulins. The isolated eel insulin produced a more pronounced and longer lasting hypoglycaemic effect than bovine insulin in the eel. Homologous eel insulin, like bovine insulin-like growth factor (IGF-I) and insulin, stimulated sulphate uptake by cultured eel cartilage in a dose-dependent manner (16-1000 ng/ml). Combination experiments using maximal concentrations of bovine IGF-I (250 ng/ml) and increasing amounts of eel insulin (10-250 ng/ml) showed no additive effects of insulin on sulphate uptake, suggesting that insulin and IGF-I may share a common mechanism(s) of action. Eel insulin and bovine IGF-I also enhanced thymidine incorporation by eel cartilage in a dose-dependent manner (4-1000 ng/ml); eel insulin was equipotent with bovine IGF-I. These results suggest that insulin, like IGF-I, may exert direct growth-promoting actions in branchial cartilage of the eel.

The complete amino acid sequence of growth hormone from sturgeon (Acipencer guldenstadti).

The complete amino acid sequence of growth hormone (GH) from a chondrostean species, the sturgeon (Acipencer gludenstaditi), has been determined. Two variants of GH, termed GH I and GH II, were isolated from the pituitary by alkaline extraction, gel filtration on a Sephadex G-100 column, and reversed-phase high-performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T column. The purified proteins were confirmed to be GHs by immunoblotting using bovine and chum salmon GH antisera. For determining of the primary structures, these GHs were digested with lysyl endopeptidase and cleaved with cyanogen bromide. The resulting fragments were separated by rpHPLC and subjected to sequence analysis on an automated gas-phase sequencer employing an Edman method. Both GHs consist of 190 amino acid residues, and contain two disulfide linkages at positions 52-163 and 180-188. The GHs differ from each other at only three positions. Sequence comparison with GHs from other vertebrates revealed that sturgeon GHs have greater sequence homology with tetrapod GHs (63-76%) than with teleost GHs (42-63%).

Isolation and characterization of glycosylated and non-glycosylated prolactins from alligator and crocodile.

Two molecular forms of prolactin (PRL), glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 M sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26,000 and 24,000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26,000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24,000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and Ile for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.

Isolation and characterization of somatolactin, a new protein related to growth hormone and prolactin from Atlantic cod (Gadus morhua) pituitary glands.

The characterization of cod somatolactin (SL), a new pituitary protein belonging to the growth hormone/prolactin family, is described. Cod SL has a molecular weight of 26 kDa and consists of 209 amino acids, of which eight are Cys. The protein has three disulfide bonds between residues Cys5-Cys15, Cys65-Cys181, and Cys198-Cys206. The Cys residues at positions 42 and 180 are not involved in disulfide bonding. The positions of these disulfide bonds are homologous to those found in prolactin and growth hormone. Cod SL has two possible N-glycosylation sites, but only one appears to have carbohydrate units attached. Chemical analysis showed the following sugars to be present: galactose, mannose, N-acetylneuramic acid, and glucosamine. A smaller variant (23 kDa) of SL has been isolated, which is believed to be deglycosylated. Sequence comparison revealed cod SL to be similarly related to both GH and PRL, but slightly higher identity was observed to the tetrapod hormones (27-33%) than to the teleost hormones (21-27%).

The complete amino acid sequences of two variants of growth hormone from Atlantic cod (Gadus morhua).

The complete amino acid sequences of two variants of cod growth hormone (GH) have been determined. The GHs, which have apparent molecular weights of 20K and 22K in SDS-PAGE, consist of 185 amino acids and have calculated molecular weights of 20,733 and 20,805, respectively. Comparison of the two sequences showed only one amino acid difference between the variants, with Lys at position 151 in the 22K GH changed to Gly in the 20K GH. The substitution of a charged amino acid by one which contains no sidechain might be expected to be reflected in the isoelectric point of the molecule. However, the observed pI for both the 20K and 22K GHs was 5.8. The difference in apparent molecular weights by SDS-PAGE suggests the existence of a conformational difference between the variants which is attributable to the observed substitution. This conclusion is in agreement with our previous data obtained from radioimmunoassay studies where the 20K GH shows only 25% cross-reactivity in an assay developed for the 22K GH. Alignment of the cod GH sequence with those of other teleost GHs reveals cod GH to be most similar to advanced marine fish such as tuna, sea bream, bonito, and yellowtail (76-83% identity), whereas it is 62-66% identical to flounder and chum salmon GH.

cDNA cloning of somatolactin, a pituitary protein related to growth hormone and prolactin.

From a flounder pituitary cDNA library, cDNA clones encoding a 28-kDa glycoprotein produced by the pars intermedia of the pituitary were isolated and characterized. Nucleotide sequencing demonstrated a precursor of the 28-kDa protein, which consisted of 231 amino acid residues, to be cleaved into a signal peptide (24 amino acids) and a mature protein (207 amino acids) containing one N-glycosylation site. By comparison of amino acid sequences, the 28-kDa protein was found to be distantly and similarly related to growth hormone and prolactin. Consequently, it was named somatolactin. Somatolactin mRNAs were specifically expressed as 1.2 and 1.8 kb poly(A)+ RNAs in flounder pituitary.

Isolation and characterization of growth hormone from a marine fish, bonito (Katsuwonus pelamis).

Growth hormone (GH) was extracted under alkaline conditions (pH 10) from pituitary glands (6.3 g) of bonito (Katsuwonus pelamis), and subsequently purified by gel filtration, ion exchange chromatography, and reversed-phase HPLC. The GH was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with yellowtail GH antiserum at each step of purification. GH activity was determined by an in vivo bioassay. The yield of this hormone was 4.8 mg/g wet tissue. Intraperitoneal injection of bonito GH at doses of 0.1 and 1 micrograms/g body wt at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. Bonito GH antiserum exhibited both species and hormone specificity in radioimmunoassay. However, the bonito GH antiserum as well as yellowtail GH antiserum exhibited hormone specificity but not species specificity in immunoblotting. A molecular weight of 21,000 and an isoelectric point of 7.0 for bonito GH were estimated by SDS-PAGE and gel electrofocusing, respectively. The complete amino acid sequence of 185 residues was determined by sequencing fragment peptides prepared by chemical and enzymatic cleavages. Sequence comparison of bonito GH with other GHs revealed that there is a significant deletion in the middle of the molecule.