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%.     

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.     

Isolation of prolactin from lyophilized porcine pituitary glands]

The optimal conditions for lyophilization of porcine pituitary glands and isolation of pure prolactin from lyophilized preparation have been investigated. The isolation method consisted in the extraction of crude pituitary preparation with acidified acetone followed by precipitation of crude prolactin preparation (acid acetone powder) by increasing the concentration of acetone in the extract to 92%. Further purification of prolactin was achieved by fractional precipitation at varying pH values and gel filtration on Sephadex G-75 column in a pH 7.5 phosphate buffer. This final procedure resulted in obtaining the monomeric form of prolactin. The identity of the isolated hormone was confirmed by spectrophotometric and radioimmunological methods as well as by polyacrylamide gel electrophoresis.     

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell)

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T₃ plasma levels in juvenile catfish.     

A selective extraction of growth hormone from bovine pituitary gland and its further purification and crystallization

A highly efficient method for the isolation of bovine growth hormone (GH) is described. The method is based on selective extraction of GH from 15,000 g subcellular sediment of anterior pituitary gland with 130-150 mM NH4HCO3, 1 mM EGTA, pH 7.2-7.4, at 2-6 degrees C for 60 min, purification of the extracted GH by ammonium sulfate fractionation, one-step ion-exchange column chromatography on DEAE-cellulose (or CM-cellulose), and gel filtration on Sephadex G-75. This 2-3 day procedure provides a highly pure hormone in high yield (up to 70-80 mg per 35-40 g of the whole pituitary gland), which can be crystallized by the batch method at a low ionic strength and isoelectric pH.     

Synthesis and secretion of phosphorylated growth hormone by rat pituitary glands in vitro

Rat anterior pituitary glands were incubated in buffered medium, pH 7.4, containing 32Pi. After incubation the tissue and medium were separated and a post-mitochondrial supernate (PMS) of the tissue homogenate was prepared. Gel filtration of the PMS and medium resulted in a radioactive peak which coincided with the elution volume of authentic rat growth hormone (rGH). Polyacrylamide gel electrophoresis of the radioactive peak under denaturing condition resulted in a protein-staining band having the same mobility as authentic rGH. Autoradiography of the gels revealed radioactivity precisely at the position of growth hormone as well as elsewhere. The specific radioactivity of the PMS [32P]GH was estimated to be 5 to 10 times greater than that of tissue [32P]GH. These results indicate that phosphorylated GH is synthesized and secreted by pituitary glands in vitro.     

Purification of carp growth hormone and cloning of the complementary DNA

The growth hormone (GH) was isolated and purified from common carp (Cyprinus carpio) pituitary glands by salt precipitation and HPLC on reverse-phase C18 columns. The carp GH cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length carp GH cDNA contains 1187 nucleotide basepairs with an open reading frame coding for the precursor form carp GH of 210 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with serine as the first residue in mature carp GH preceded by a 22-residue hydrophobic signal peptide. Comparison of the amino-acid sequence of carp GH with those of various species reveals positional identity at 32.4%, 38.8%, 42.0%, 37.2%, 66%, 55% and 49% with GHs of man, rat, duck, bullfrog, salmon, tuna and yellow tail, respectively.     

Application of artificial gel antibodies for investigating molecular polymorphisms of human pituitary growth hormone

Artificial gel antibodies were used to investigate human growth hormone (GH) activity in preparations purified from human pituitary glands. A partially purified fraction containing differently sized structural variants of GH was processed to yield monomeric and dimeric forms suitable for synthesizing artificial polyacrylamide gel antibodies. These two types of GH antibodies were used for investigating GH activity in experiments using HPLC gel-permeation and ion-exchange chromatography. In the size-exclusion experiments, both hormone fractions eluted as homogeneous peaks, whereas the ion exchanger resolved the hormones into several active components. The GH monomer antibodies exhibited a much higher affinity for monomeric GH than for dimeric GH, and the GH dimer antibodies exhibited a much higher affinity for dimeric GH than for monomeric GH. It was concluded that these two sets of antibodies might be useful for discriminating between dimeric and monomeric GH in clinical samples.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Intracellular location of newly synthesized growth hormone

The current model for the synthesis and secretion of protein hormones by cells of the anterior pituitary leads to a prediction that newly synthesized GH should be membrane-associated. Experiments were performed to test this prediction. Rat pituitary tumor cells (GH₃) were pulsed with ³H-leucine for 2 min, and chased with cold leucine for min. Analysis of subcellular fractions by SDS polyacrylamide gel electrophoresis and by immunoprecipitation of GH demonstrated that most (86%) of the newly synthesized GH is membrane-associated.     

Electrophoretic separation of forms of rat growth hormone with different bioassay and radioimmunoassay activities: comparison of intraglandular and secreted forms

Rat adenohypophysial tissue and medium in which pituitary explants had been incubated were processed by polyacrylamide disc gel electrophoresis (pH 9.5). Eluates of 6 regions of the gel columns were tested for growth hormone (GH) by a bioassay (BA; rat tibia test) and a radioimmunoassay (RIA). In the tissue, both assays detected most of the GH (80–99%) in segments with an R_f range of 0.30–0.56. In the medium, both assays recorded a more cathodal distribution of most of the hormone (85–96%) in the R_f region 0.00–0.42. The BA/RIA ratios of the segment containing the prominent stainable GH band (R_f range 0.30–0.42) varied from 0.6–1.8 in tissue and medium. The tissue contained slow migrating (R_f range 0–0.29) GH that had a high BA/RIA ratio (11.1–25.7). The medium contained high ratio GH (32.7–33.8) in a more anodal location (R_f 0.57–0.68). Purified rat prolactin (RP-I) was not active in either the BA or the RIA. The results indicate that rat GH exists in electrophoretically heterogeneous forms which differ in their BA/RIA activity ratios. The proportion of these forms stored in adenohypophysial tissue and secreted by the gland may differ.     

Purification and properties of bovine pituitary follitropin.

A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.     

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells. (J Histochem Cytochem 58:881–889, 2010)     

Purification of lutropin and follitropin in high yield from horse pituitary glands

A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitaries, respectively. Subunits were prepared by dissociation in 8 M guanidine HCl followed by either gel filtration (eLH) or gel filtration followed by QAE-Sephadex chromatography (eFSH). The hormones and their subunits were characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, NH2-terminal analysis, and by LH and FSH radioligand receptor assays.     

Prostaglandin E2 (PGE2)-induced growth hormone (GH) release: Effect of intrahypothalamic and intrapituitary implants

Overiectomized rats were unilaterally implanted with a 23-gauge stainless steel cannula in different hypothalamic areas or in the pituitary gland and subsequently were treated with estrogen (sc, 10 μg estradiol benzoate, Eb). Two days after the estrogen injection, an inner cannula containing PGE₂ or PHF_2α at its tip was inserted into the cannula. Other animals were implanted with empty inner cannula. Plasma GH concentrations were measured by RIA in blood samples drawn from the jugular vein while the animals were lightly etherized before (−2) and at 20, 40, 60 and 120 min following the implantation. Plasma GH levels in control animals bearing an empty cannula in the body of the arcuate nucleus-median eminence region (BARH-ME) were signifantly depressed by the ether stress. The implantation of PGF_2α in this area was completely ineffective in preventing ether stress-induced decline in plasma GH. By contrast, PGE₂ implanted in BARH-ME or the post-chiasmatic region of the hypothalamus (HARH-ME) elevated plasma GH 20 min following its implantation and partially prevented the subsequent decrease in GH levels induced by ether stress. PGE₂ implants located in several other hypothalamic areas failed to induce GH release or to prevent the decline in GH levels induced by ether stress. However, PGE₂ implanted in the pituitary gland elicited a marked increase in plasma GH at 20 min and completely prevented the subsequent ether stress-induced decline in GH levels.The results suggest that PGE₂ can act at both hypothalamic (ARH-ME) and pituitary levels to stimulate GH release. At the hypothalamus, PGE₂ may inhibit GH-inhibiting factor (GIF) release or induce release of GH releasing factor (GHRF).     

大鳞大马哈鱼垂体生长激素的分离、纯化与鉴定

应用ConA-Sepharose 4B亲和层析、凝胶过滤及离子交换层析等技术从大鳞大马哈鱼(Oncorhynchus tshawytscha)垂体中分离纯化了具有生物活性的生长激素(sGH)。用放射受体测定法(RRA)检测sGH组分的生物活性,结果表明纯化的8GH制品具有与兔肝细胞GH受体结合的生物活性。用放射免疫测定法(RIA)和酶联免疫吸附测定法(ELISA)分别检测了另两种垂体激素-催乳激素(PRL)和促性腺激素(GTH)在纯化的sGH制品中的残留量均在0.5％以下。用SDS-聚丙烯酰胺凝胶电泳SDS-PAGE评价sGH制品的电泳纯度并测定了其分子量为22000左右。等电聚焦电泳表明该种鱼GH由等电点分别为6.3和6.6的两种形式的分子组成。     

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands.

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.