Detection of multiple PRL- and GH-like proteins in human pituitary by Western blot analysis

Surgically removed normal and tumorous pituitary tissues from a prolactinoma patient were analyzed by Western blot techniques for PRL and GH variants. Criteria for identification were the Rf of the bands within the gel, immunologic crossreactivity to specific antisera, and structural verification by tyrosine peptide-mapping of individual bands from the gel. The authors found the tumor-tissue to be characterized by the presence of a PRL band greater in concentration than in the normal tissue and the virtual absence of a GH band. Immunoblotting of the electrophoretically resolved proteins form both types of tissues revealed several new bands crossreactive with human PRL and GH antibodies. Some of the new bands were of Mr greater than the monomeric PRL and GH and others were of lower Mr. Relative of two of the low mobility Mr PRL-immunoreactive bands designated as 16K and 8K corresponded to the Rf of the two fragments of cleaved PRL, which suggested that cleaved PRL occurred naturally in the human pituitary gland. The most conspicuous of the new PRL-immunoreactive bands, a 25,000 Mr protein migrating slightly behind PRL, displayed strong crossreactivity to hPRL antibodies and was present in greater concentration in the prolactinoma tissue than in the normal tissue. These properties suggested that it was related to hPRL and perhaps represented its glycosylated variant. However, its tyrosine peptide map did not resemble that of hPRL. Thus, it is not clear whether it represented G-hPRL or a new pituitary protein that cross-reacts with hPRL antibodies. In addition, two other bands of low Mr, designated as unknown 1 and unknown 2, reacted with hPRL antibodies. Immunostaining with hGH antibodies revealed the 20K-hGH variant, the F1 fragment of cleaved hGH, and a pair of new bands immediately behind GH that could represent glycosylated hGH--possibly a product of Seeburg's variant hGH gene. Both PRL and GH antibodies elicited numerous bands of high Mr by the technique employed, far more than ever observed by Sephadex chromatography. The nature of the high Mr bands remains unknown. Further characterization of these new PRL- and GH-immunoreactive proteins might help in the understanding of the multiple physiologic functions of PRL and GH in man.     

Isolation and characterization of growth hormone from Atlantic cod (Gadus morhua)

Growth hormone was purified from cod pituitary extract by a simple two-step procedure involving gel filtration and reversed-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by rpHPLC, SDS-polyacrylamide gel electrophoresis, and immunoblotting using anti-chum salmon growth hormone (GH) antiserum. The yield of purified hormone was 1.3 mg/g pituitary. Cod GH was found to exist in two monomeric forms (Mr = 20K and 22K) and dimeric forms (Mr = 40K and 42K). The two monomeric forms have a pI of 5.8, an identical amino acid composition, histidine as the N-terminal residue, and an identical lysyl endopeptidase peptide map. Staining with concanavalin A was observed on the 20K component only, but analysis for total reducing sugar did not confirm these results. Cod GH was found to be a potent stimulator of growth in juvenile rainbow trout which received intraperitoneal injections of the hormone. The partial amino acid sequence has been determined.     

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.     

Further characterization of growth hormone from the chum salmon (Oncorhynchus keta)

This report describes the isolation of growth hormone (GH) from the chum salmon (Oncorhynchus keta) pituitary using gel, affinity, and ion exchange chromatography. Chum GH has an estimated molecular weight of 23,500 and an amino acid composition that is consistent with a vertebrate GH. The differentially charged forms of chum GH which are only apparent under alkaline conditions were separated by ion exchange and compared immunologically and biologically; Peak I, which consists of a single band (Rf = 0.35) under alkaline electrophoresis and Peak II which consists of two bands with Rf's of 0.41 and 0.45. Both forms were found to be immunologically identical by immunodiffusion and to have similar growth promoting properties in intact rainbow trout (Salmo gairdneri). Chum GH was also active in the rat tibia test at a daily dosage of 70 micrograms/animal. The results are discussed in relation to previous studies with chum GH and other fish GHs.     

Purification and properties of teleost growth hormone

Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedimentation coefficient (s_20,w) of 2.19, and an α-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.     

Greater conservation of somatolactin, a presumed pituitary hormone of the growth hormone/prolactin family, than of growth hormone in teleost fish

From pituitary cDNA libraries of Atlantic cod and chum salmon, cDNA clones coding for somatolactin (SL), a presumed pituitary hormone belonging to the growth hormone (GH)/prolactin (PRL) family, were isolated and characterized. The 1.3-kb cod SL mRNA was composed of a greater than 0.25-kb 5' untranslated region, a coding region for the precursor of 235 amino acids (aa), a 0.14-kb 3' untranslated region, and a poly(A) tail. The 2.5-kb salmon SL mRNA had a less than 0.1-kb 5' untranslated region, a precursor (233 aa) coding region, a 1.6-kb 3' untranslated region, and a poly(A) tail. A signal peptide of 26 and 24 aa was found in the SL precursor of cod and salmon, respectively. Thus, the mature SLs of these fish are composed of 209 aa. Two potential N-glycosylation sites were identified in cod SL, whereas no site could be found in the salmon. A comparison of amino acid sequences of the three SLs so far isolated indicated six Cys residues to be in homologous positions to those in GH and PRL, and one Cys residue to be characteristically present in SL. Among cod, salmon, and flounder, greater colinearity of amino acid sequences was noted in SLs than in GHs. The identities of the SL amino acid sequences were between 73 and 81% as compared to 58-62% for the corresponding GHs, indicating greater conservation of SL than GH.     

The primary structures of growth hormones of three cyprinid species: bighead carp, silver carp, and grass carp.

The primary structures of growth hormone (GH) of three cyprinid species, bighead carp, silver carp, and grass carp, were determined by a chemical method and/or by molecular cloning. The bighead carp GH was extracted from pituitary tissue by use of an alkaline medium, then successively purified by gel filtration, hydrophobic interaction column chromatography, and reverse-phase high-pressure liquid chromatography. The primary structure of bighead carp GH determined chemically is identical to that deduced from the cDNA nucleotide sequence. By molecular cloning, the primary structures of silver carp and grass carp GHs were also determined. The GHs of these three cyprinid species all contain 188 amino acid residues and their sequences are identical. When four of the five cysteines of cyprinid GHs were arranged to match the same positions of cysteines of other vertebrate GHs, a maximally matched alignment was achieved. Among fishes, GHs are relatively conserved within the same order (82 to 100% identity) but they are more diversified between orders (49 to 68% identity). In further comparison, fish GHs are even more different from tetrapod GHs (37 to 58% identity). Although the primary structures of vertebrate GHs are relatively variable, four homologous sequences, notably one located at the C-terminal, are found.     

The primary structure of coho salmon growth hormone and its cDNA

Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3'-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.     

The metabolic clearance, distribution, and degradation of dimeric and monomeric growth hormone (GH): implications for the pattern of circulating GH forms

The ratio of oligomeric (big) to monomeric (little) human (h)GH forms in plasma exceeds that in the pituitary gland severalfold. To investigate whether delayed metabolic clearance of oligomers could explain this discrepancy, we measured MCR, distribution volumes, and degradation rates of radio-labeled hGH22K dimer, hGH20K dimer, hGH22K monomer, and hGH20K monomer in the rat. Hormones were injected as a bolus, and disappearance from plasma was followed by immunoprecipitation and trichloroacetic acid precipitation. MCRs of the dimers were significantly lower than those of the corresponding monomers (5-fold in the case of hGH22K, and 2-fold in the case of hGH20K). Both dimers were also degraded at slower rates than the monomers. Distribution volumes for the dimers, although somewhat smaller, were not statistically different from those for the monomers and were consistent with distribution in the extracellular space. We conclude that hGH dimers are relatively protected from degradation and hence cleared more slowly from the blood than hGH monomers. This may lead to their accumulation in the circulation relative to their monomeric counterparts, which may explain their high proportion in plasma as compared to pituitary.     

Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone

We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.     

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.     

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.     

Immunoaffinity purification and partial characterization of sea bass (Dicentrarchus labrax) growth hormone

Growth hormone (GH) was isolated from sea bass (Dicentrarchus labrax) pituitary extract by a simple one-step procedure involving immunoaffinity chromatography. A monoclonal antibody raised against chicken GH and found to immunostain very specifically the GH cells in the pituitary of the sea bass was coupled to CNBr-activated Sepharose 4B. Sea bass pituitary extracts were run on the affinity column, and the eluted material was analyzed on reversed-phase HPLC and found to consist of one single peak. The yield of purified hormone was 2.4 mg/g pituitary. Two monomeric forms (MW = 20,000 and 22,000 Da) of sea bass GH were identified by gel electrophoresis. Gel electrofocusing revealed apparent isoelectric points of 6.15, 6.50, and 6.95. Amino acid composition is consistent with other vertebrate GHs. The immunological relatedness was tested by immunoblotting using antisera raised against GH of different species. Polyclonal antisera raised against the isolated hormone exhibited a specific labeling of the GH cells in sea bass pituitary sections as well as of the immunoblotted purified GH.     

Immunochemical and biological studies with growth hormone in a pituitary extract of the coelacanth, Latimeria chalumnae Smith.

A substance immunochemically related to GH has been demonstrated in an extract of a pituitary from a coelacanth which is considered to be the sole surviving member of the ancient Crossopterygians. The findings were based on studies employing radioimmunoassay with an antiserum to turtle GH which was shown recently to be capable of detecting immunochemical relatedness of purified GHs from various vertebrate species with respect to those mammals. The results demonstrated that the coelacanth extract does contain a substance which shows a low, but significant degree of relatedness to GHs of tetrapods and to a substance immunochemically related to GHs in pituitary extracts of other existing primitive fishes including the lungfish, shark and sturgeon. Bioassay of the pituitary extract revealed that it was capable of a low, but significant degree of stimulation of growth promotion in a mammal on the basis of the rat tibia assay. The significance of the results is discussed.     

The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant

This work was undertaken to study the heterogeneity of GH in serum and placental and pituitary extracts and to study GH physiology in pregnant women. Two distinct monoclonal antihuman GH (anti-hGH) antibodies (MAb) coded 5B4 and K24 were selected for their high binding affinity and specificity. The 5B4 MAb recognized the epitope comprising the NH2-terminal end of hGH, and the K24 MAb recognized an internal epitope. Both MAbs were used in RIAs to measure serum GH concentrations in various circumstances, including pregnancy. The two RIAs yielded slightly different serum GH results in normal men and nonpregnant women, but the overall correlation between the data was excellent. Since the RIAs were not affected by human placental lactogen, the evolution of serum GH in pregnant women could be studied. In such women, serum GH levels progressively declined to undetectable levels during the second half of pregnancy, while a pregnancy-associated serum GH-like antigen [tentatively called human placental growth hormone (PGH)] appeared in the circulation at midpregnancy and increased thereafter up to term. PGH contained the NH2-terminal epitope of pituitary GH, but lacked the internal one. Consequently, it reacted selectively with the 5B4 MAb only. After delivery, PGH disappeared from maternal serum within 1 h. Amniotic fluid contained low GH concentrations; cord serum contained high GH levels, but no PGH. Thus, PGH appears to be secreted selectively into the maternal compartment. PGH was purified from term placenta extracts. According to its chromatographic behavior, it appears more basic than pituitary 22K and 20K GHs. Size dimorphism was demonstrated; PGH was composed of two entities of 22K and 25K, respectively. Pure PGH, obtained in small quantities by preparative electrophoresis, was found to bind to hepatic GH receptor with an apparent high potency compared to that of pituitary GH, PGH, thus, should act in vivo as a GH agonist sharing most of its biological properties. These results lead to the conclusion that PGH is likely to replace the pituitary hormone in governing maternal metabolism during the second half of pregnancy.     

Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization

Multiple growth hormone (GH)-binding proteins (GHBPs) were identified in serum and plasma samples from domestic chickens and turkeys. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% acrylamide, 2.7% bis discontinuous gels under reducing conditions and electrotransferred to nitrocellulose paper. Western blots were incubated with 125I-labelled recombinant chicken GH (cGH) or bovine GH and GHBPs visualized by means of autoradiography. In fresh samples (less than 2 h from collection to gel electrophoresis), multiple minor high Mr bands were evident between approximately 72,000 and 175,000. Two major bands were observed at approximately 69,500 and 27,500. The latter is consistent with previous reports for the rat and mouse serum GHBPs based on nucleotide sequence analysis. The minor bands were essentially undetectable after storage at -25 degrees C for several months, and an additional major band at Mr approximately 52,500 appeared. The Mr-69,500 major protein contained N-linked carbohydrate, as determined by a reduction in molecular size by treatment with peptide N-glycosidase F. Binding of 125I-labelled GH was partially inhibited by co-incubation with 50 micrograms unlabelled pituitary-derived cGH/ml and excess unlabelled porcine GH as well as ovine prolactin, but not by bovine insulin. Non-specific binding of 125I-labelled GH by serum albumin was also observed. A comparison was made between these GHBPs and the hepatic GH receptor (e.g. molecular weight estimates, affinity for homologous versus heterologous GHs, cross-reactivity with prolactin, presence of N-linked carbohydrate). The origin and relationship among the various molecular weight species of GHBPs identified, and their potential role in regulation of the biological activity of GH in birds, remain to be determined.     

Hamster growth hormone. Species specificity and physiological changes in blood and pituitary concentrations as measured by a homologous radioimmunoassay

A specific and sensitive homologous radioimmunoassay (RIA) for determination of golden hamster growth hormone (GH) is described and compared to a heterologous hamster GH RIA. Using the homologous system, cross-reactivity experiments between golden hamster GH on one hand, and pituitary extracts from two other hamster species, and purified GHs from several rodent and mammalian species, suggested that hamster GH differs from other rodent and mammalian GHs to a greater extent than rat GH. Using the homologous method, we have determined that, in the golden hamster, diurnal plasma fluctuations have a mean interpeak interval of 70-80 min, that serum GH concentrations are affected by nyctohemeral differences in lighting, by ether vapors, by animal's gender, and by starvation and refeeding. Basal GH concentrations were not influenced by exposure to different photoperiods or by removal of the gonads. A heterologous hamster GH RIA, which utilizes a radioiodinated rat GH and monkey antihamster GH serum, represents the most advantageous method for the measurement of hamster GH because of its high sensitivity (0.3 ng/ml), low limit of detection (46 pg/ml), high antiserum binding to iodinated rat GH, and the ability to measure rat and mouse GHs in addition to hamster GH. Immunological differences between hamster, rodent, and other mammalian GHs are diminished when heterologous, rather than homologous, tracer is used.     

Slow metabolic clearance rate of the 20,000-dalton variant of human growth hormone: implications for biological activity

The 20,000-dalton variant of human GH (hGH) (20 K) exhibits full growth-promoting bioactivity in the rat despite its poor interaction with GH receptors, as compared to the principal 22,000-dalton form of hGH (22 K). To test the possibility that prolonged survival time of 20 K in vivo may contribute to this apparent discrepancy, we compared the MCRs of 22 K and 20 K in the rat by the single injection technique. Both radiolabeled and native 22 K and 20 K were examined in this regard. The MCR of 20 K was 2- to 3-fold lower than that of 22 K, a statistically significant difference (P less than 0.05). The distribution volumes were similar for the two hGH forms and corresponded approximately to the extracellular fluid space. We conclude that the prolonged persistence of 20 K in the circulation may contribute to its higher than expected bioactivity in vivo in the rat.     

Phosphorylation of prolactin and growth hormone.

To determine whether GH and prolactin could be phosphorylated, turkey GH, chicken GH, chicken prolactin and turkey prolactin were incubated in vitro with the catalytic subunit of protein kinase A and [gamma-32P]ATP. Phosphorylation was assessed after sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and autoradiography. Polyacrylamide electrophoresis showed that both purified native chicken GH and turkey GH were phosphorylated under the conditions employed. However, the glycosylated variant of chicken GH did not appear to be labelled. Chicken prolactin, turkey prolactin and the glycosylated variant of turkey prolactin were all intensely phosphorylated by protein kinase A. Ovine and rat prolactins could also be phosphorylated by protein kinase A. The phosphate content of different native prolactin (turkey, ovine and rat) and GH (ovine and chicken) preparations was also determined and found to be significant. Chicken pituitary cells in primary culture incorporated 32P in GH- and prolactin-like bands isolated by non-denaturing polyacrylamide gel electrophoresis, and this was stimulated by phorbol myristate acetate. Phosphorylation of GH and prolactin may thus explain some of the charge heterogeneity of these hormones.     

Isolation of sockeye salmon growth hormone utilizing serum triiodothyronine enhancement in rainbow trout to monitor biological activity.

Growth hormone (GH) was isolated from sockeye salmon (Oncorhynchus nerka) pituitary glands using established techniques of affinity and gel filtration chromatography, and preparative polyacrylamide gel electrophoresis. The GH activity was followed throughout the fractionation procedure with a bioassay based on the increase of serum triiodothyronine (T3) in rainbow trout (Oncorhynchus mykiss), measured by radioimmunoassay (RIA). Amino-terminal amino acid sequence analysis and subsequent comparison with established GH sequences from other Oncorhynchus sp. were used to confirm the isolation of sockeye salmon GH (ssGH). The bioassay was sensitive to a dose of 55 ng of purified ssGH/g fish. Monomeric GHs, located in the carbohydrate-poor protein fraction, were the only pituitary components that elevated serum T3. Twenty-four hours after GH injection was an appropriate and practical time to blood sample, allowing completion of the bioassay, including RIA, in 3 days. The generic homology, between the source of pituitaries and the bioassay animals used in this study, should permit the bioassay to be useful during GH isolation from pituitaries of all Oncorhynchus sp.