Kazuo Yamaguchi

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.

Isolation of two forms of growth hormone secreted from eel pituitaries in vitro

Two forms of growth hormone (GH) were purified by chromatofocusing of medium from cultured Japanese eel (Anguilla japonica) pituitaries. The pituitaries were organ-cultured in Eagles minimum essential medium with Earles salts. Following polyacrylamide gel electrophoresis of the medium at pH 9.5, two prominent bands were seen with Rf 0.36 and 0.29; they were designated as eGHI and eGHII, respectively. Seven-hundred fifty milliliters of medium, in which 260 pituitaries were cultured for 6-10 weeks, was concentrated by DIAFLO membrane (YM-5) and subjected to gel filtration on a Sephadex G-75 column and to chromatofocusing on a PBE-94 column. eGHI and II were finally purified by gel filtration on a Sephadex G-75 column, yielding 2.0 mg of eGHI and 1.3 mg of eGHII. Both eGHI and eGHII were equipotent to ovine GH in promoting growth of juvenile rainbow trout. The putative GH-producing cells in the proximal pars distalis of the eel pituitary were stained specifically with antisera raised against eGHI or eGHII; no cross-reactivity was seen in the follicular prolactin cells in the rostral pars distalis. As determined by gel isoelectric focusing, eGHI and eGHII have isoelectric points of 6.3 and 6.7, respectively. Identical molecular masses of 23,000 Da were determined by sodium dodecyl sulfate gel electrophoresis. Their amino acid compositions strongly resembled each other; comparison of the partial N-terminal amino acids indicates that sequence 1 to 36 of GHII is exactly the same as 4 to 39 of GHI.

Primary structure of common carp prolactins

The complete amino acid sequence of common carp (Cyprinus carpio) prolactin (PRL) has been determined. One milligram of carp PRL was reduced, carboxymethylated, and then cleaved with cyanogen bromide and several enzymes. Another milligram of carp PRL was digested with lysyl endopeptidase. The resulting fragments were separated by reverse-phase high-performance liquid chromatography and subjected to sequence analysis by the automatic Edman method with a gas-liquid sequencer and the manual DNS-Edman method. The carp PRL consists of 186 amino acid residues with two disulfide linkages formed between residues 46-160 and 177-186. In addition, it lacks the linkage in the amino-terminal portion of mammalian prolactins, as do salmon and tilapia PRLs, and shows 77% sequence identity with salmon PRL and 36% identity with mammalian PRLs. The sequence comparison has been performed with 190 alignment positions. Four conserved segments of the alignment positions 1-32, 46-66, 71-94, and 155-182 that were found between salmon and mammalian PRLs are further confirmed in carp PRL. Variant residues are clustered in the region of residues 98-143 which has 48% sequence identity with salmon PRL and only 2% with mammalian PRL.

Primary structure of Eel (Anguilla japonica) growth hormone

Two molecular forms of growth hormone, GHs I and II, were isolated from a culture medium of the eel pituitary (Anguilla japonica). The complete amino acid sequence of GH I was determined in the present study. The hormone was reduced, carboxymethylated, and subsequently cleaved with cyanogen bromide and enzymes. Intact eel GH I was also digested with enzyme. The resulting fragments were separated by reverse-phase high-performance liquid chromatography (HPLC) and subjected to sequence analysis by automated gas-liquid sequencer employing an Edman method. Eel GH I consists of 190 amino acid residues with two disulfide linkages formed between residues 52-163 and 180-188. Sequence comparison with other vertebrate GHs revealed that eel GH I is closer to avian and mammalian GHs with 55% identity than to salmon GH with 48% identity.

The complete amino acid sequence of growth hormone of an elasmobranch, the blue shark (Prionace glauca).

The complete amino acid sequence of growth hormone (GH) from a phylogenetically ancient fish, the blue shark (Prionace glauca), was determined. The shark GH isolated from pituitary glands by U. J. Lewis, R. N. P. Singh, B. K. Seavey, R. Lasker, and G. E. Pickford (1972, Fish. Bull. 70, 933-939) was purified by reversed-phase high-performance liquid chromatography. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting peptide fragments were separated by rpHPLC and submitted to sequence analysis by automated and manual Edman methods. The shark GH consists of 183 amino acid residues with a calculated molecular weight of 21,081. Sequence comparisons revealed that the elasmobranch GH is considerably more similar to tetrapod GHs (e.g., 68% identity with sea turtle GH, 63% with chicken GH, and 58% with ovine GH) than teleostean GHs (e.g., 38% identities with salmon GH and 42% with bonito GH) except for eel GH (61% identity), and substantiates the earlier finding derived from the immunochemical and biological studies (Hayashida and Lewis, 1978) that the primitive fish are less diverged from the main line of vertebrate evolution leading to the tetrapod than are the modern bony fish.

The complete amino acid sequence of growth hormone from the sea turtle (Chelonia mydas).

The complete amino acid sequence of growth hormone (GH) from a reptilian species (the sea turtle, Chelonia mydas) has been determined for the first time. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting fragments were exclusively separated by reversed-phase high-performance liquid chromatography and subjected to sequence analysis by automated gas-phase sequencer employing the Edman method. The sea turtle GH consist of 190 amino acid residues with two disulfide linkages formed between residues 52-160 and 180-188, and possesses a microheterogeneity, indicated by the presence or absence of an additional alanine residue at the N-terminus. Sequence identities of sea turtle GH to other species of GH are 89% with chicken GH, 79% with rat GH, 68% with blue shark GH, 58% with eel GH, 59% with human GH, and 40% with a teleostean GH such as chum salmon. On the basis of amino acid sequence comparisons, a molecular phylogenetic tree is proposed.

Purification and characterization of bullfrog growth hormone

A highly purified growth hormone (GH) was isolated from an unadsorbed fraction obtained by subjecting acid acetone extract of bullfrog pituitary glands to DEAE-cellulose column chromatography, a side fraction obtained during the purification of prolactin, by cation-exchange chromatography on CM-Toyopearl and high-performance liquid chromatography on ODS with a yield of 5.6 mg/g protein of the starting material. Intraperitoneal injections of GH to hypophysectomized Xenopus resulted in a considerable elevation of chondroitin sulfate synthesis in the xiphisternal cartilage as measured in vitro. The bullfrog GH had a molecular weight of 22,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The isoelectric point of bullfrog GH was estimated to be 7.8 by gel electrofocusing. The partial amino acid sequences of bullfrog GH at both terminal regions were determined.

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%).

The complete amino acid sequence of prolactin from the bullfrog, Rana catesbeiana

The complete amino acid sequence of prolactin (PRL) from an amphibian species, the bullfrog (Rana catesbeiana), has been determined and conserved residues and domains were analyzed by sequence comparison of PRLs from 15 species of five major vertebrate classes. The bullfrog PRL consists of 197 amino acid residues with three disulfide linkages formed between residues 4-11, 58-172, and 189-197. The bullfrog PRL shows the highest identity with sea turtle PRL (75%); lower identities with chicken PRL (72%), pig, horse, and fin whale PRLs (68%), human, cattle, sheep, and elephant PRLs (60-58%), and rat and mouse PRLs (50%); and significantly lower identity with teleost PRLs (about 30%). It is apparent that all tetrapod PRLs characterized so far contain three disulfide bonds in homologous positions and differ from teleost PRLs which lack the N-terminal disulfide loop. The tetrapod and teleost PRLs share 34 common residues and these conserved residues are clustered in six domains (PD1 to PD6), suggesting that these common residues, or at least part of them, are responsible for the activities common to all PRLs. On the other hand, PD5 is conserved significantly within tetrapod PRLs, but to a lesser extent in teleost PRLs, suggesting that the PD5 contributes to the activities specific to tetrapod PRLs.

Amino acid sequence of growth hormone isolated from medium of incubated pituitary glands of tilapia (Oreochromis mossambicus)

The amino acid sequence of tilapia (Oreochromis mossambicus) growth hormone (GH) was determined directly by Edman degradation of peptide fragments generated by lysyl endopeptidase and trypsin digestion. The N-terminal residue was deduced to be pyroglutamic acid through the use of pyroglutamyl aminopeptidase; its removal allowed amino acid sequence determination of the remainder of the N-terminal trypsin peptide by Edman degradation. Tilapia GH is composed of 187 amino acid residues and shows high similarity to other perciform GHs. Sequence identities are: 89% with tuna GH, 83% with bonito GH, 85% with yellowtail GH, 89% with red sea bream GH, and 34% with bovine GH. The two asparagine residues (Asn-148 and Asn-184) were recovered by Edman degradation, suggesting the absence of N-glycosylation.