Purification and primary structure of glucagon from ostrich pancreas splenic lobes

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

Biochemical isolation and physiological identification of the egg- laying hormone in Aplysia californica

It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone.     

The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus.

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.     

Isolation, characterization and opiate activity of beta-endorphin from the pituitary gland of the ostrich, Struthio camelus.

1. Avian beta-endorphin was purified from adenohypophyseal glands of the ostrich Struthio camelus by a procedure involving acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography, Sephadex G-50 chromatography and paper electrophoresis (pH 6.7). 2. The 31-amino acid peptide behaved as a single substance during polyacrylamide-gel electrophoresis and isoelectric focusing, the isoelectric point being 8.84. 3. Ostrich beta-endorphin exhibited significant opiate activity in the guinea-pig ileum preparation.     

Changes in the isoelectric focusing profile of pituitary follicle-stimulating hormone in the developing male rat

Pituitary glands, hypothalami, and trunk blood were obtained from male rats at 5, 15, 18, 21, and 29 days of age, on the day of balanopreputial separation (Days 42-45), and during adulthood. The forms of follicle-stimulating hormone (FSH) present within each pituitary were separated by polyacrylamide gel isoelectric focusing. Serum and pituitary gonadotropins, hypothalamic luteinizing hormone-releasing hormone (LHRH), and the profile of FSH forms across the isoelectric focusing gel were determined by radioimmunoassay. No change in the relative proportions of FSH forms were observed between 5 and 21 days of age. Likewise, only slight changes in serum and pituitary gonadotropin levels and hypothalamic LHRH content were observed at these times. After 21 days of age, dramatic increases in serum and pituitary gonadotropin levels were observed. Similarly, a shift in FSH forms within the pituitary to more basic and bioactive forms was observed at this time. These results demonstrate that, during the transition through puberty in the male rat, not only the absolute amount, but also the isoelectric focusing profile, of FSH change.     

Adrenocorticotropin. 57. Synthesis and biological activity of ostrich and turkey hormones

Synthesis of ostrich and turkey corticotropin (ACTH) has been accomplished by the solid-phase method. Each was identical to the natural hormone in high performance liquid chromatography. Relative potencies in a lipolytic assay in isolated rabbit fat cells were: human ACTH, 100; ostrich ACTH, 53; turkey ACTH, 28. In isolated rat fat cells relative lipolytic potencies were: human ACTH, 100; ostrich ACTH, 2; turkey ACTH, 13. It was concluded that lipolytic potency is sensitive to alterations in structure throughout the entire length of the ACTH sequence in the rat fat cell assay.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A280/A260 less than 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.     

Recombinant-DNA-derived bovine growth hormone from Escherichia coli. 2. Biochemical, biophysical, immunological and biological comparison with the pituitary hormone

Bacterially synthesized, recombinant-DNA-derived bovine growth hormone (r-bGH), prepared as described in the preceding paper in this journal, has been characterized in comparison with pituitary bovine growth hormone (pit-bGH). The characterization criteria include sodium dodecyl sulfate/polyacrylamide gel electrophoresis, automated N-terminal sequence analysis, amino acid composition, isoelectric focusing, reverse-phase high-performance liquid chromatography, ultraviolet absorbance, analysis for free protein thiol, sizing by gel filtration, circular dichroism, radioimmunoassay and biological activity in the hypophysectomized rat weight-gain assay. In every respect the r-bGH appears to be virtually identical to pit-bGH.     

Osteoinductivity of partially purified native ostrich (Struthio camelus) bone morphogenetic protein: comparison with mammalian species

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. They are capable of inducing ectopic bone formation. Until now, the main interest has been focused on mammalian osteoinductive BMPs, and there are no reports of native BMP extracts of birds. In this study, we isolated and characterized native BMPs of ostrich (Struthio camelus) and compared them with identically isolated native bovine (cow) and reindeer BMPs with regard to BMP pattern and osteoinductive capacity. The ostrich BMP pattern differed markedly from that of cow and reindeer BMP in non-reduced SDS-PAGE, reduced SDS-PAGE and Western blot. The differences in isoelectric focusing analysis were smaller. However, the ostrich BMP extract had a peak at pH 5.1, clearly differing from the BMPs of cow and reindeer. The osteoinductive capacity and density of ectopic bone, induced by BMP extracts in a mouse thigh muscle pouch, were determined radiographically. The ostrich BMP extract displayed significantly lower osteoinductive capacity and density of induced bone than the bovine and reindeer BMP extracts. In conclusion, our results indicate that the BMP pattern of birds differs considerably from that of mammals, and that the osteoinductive capacity of BMPs and the density of induced bone are lower in birds than in mammals. They also suggest that the bone metabolism of birds is adapted to make light bones suitable for flying.     

鲈鱼生长激素的分离及其生物活性鉴定

运用葡聚糖凝胶Ｇ－１００过滤和反相高儿液相色谱纯化两步法,首镒从鲈鱼脑垂体中分离出鲈鱼生长激素,通过ＳＤＳ－聚丙烯凝胶电泳测得鲈鱼非还原性的和还原性的生长激素分子量分别为１９．２和２０．７ｋＤ;等电聚焦证实鲈鱼生长激素等电点为７．１５。Ｗｅｓｔｅｒｎ免疫印迹反应证实,鲈鱼生长激素具有与大麻哈鱼生长激素抗体发生特异性免疫交叉反应的特性,而与大麻哈鱼催乳素和生长催乳素抗体无免疫交叉反应。     

A carrier lipoprotein for juvenile hormone in the haemolymph of Locusta migratoria

In the haemolymph of adult female locusts six different lipoprotein fractions have been demonstrated by means of isoelectric focusing. One of these binds injected ³H-Cecropia juvenile hormone. The carrier protein is a yellow lipoprotein with a molecular weight of approximately 220,000 daltons and an isoelectric point of pH 6·8. The binding of the hormone to the protein is stable during gel filtration over Sephadex G-25 and during dialysis for 24 hr against phosphate buffer pH 7·0.The hormone is quickly metabolized in the locusts. In the haemolymph were found more polar compounds such as 10-epoxy-7-ethyl-3,11 dimethyl-2,6-tridecadienoic acid and the corresponding dioldienoic acid.Both compounds were not bound by the pH 6·8 carrier lipoprotein under in vivo conditions.     

The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.     

Purification and characterization of chicken follicle-stimulating hormone.

1. Highly purified chicken follicle-stimulating hormone (cFSH) was isolated from chicken pituitaries by differential extraction, sequential chromatography on HPLC cation and anion exchange columns, and gel filtration chromatography. 2. Purified cFSH (USDA-cFSH-K-1) had a potency of 77.44 units/mg in a chicken testes radioreceptor assay, and was biologically active in stimulating the secretion of progesterone by chicken granulosa cells. 3. Purified cFSH contained negligible luteinizing hormone and thyroid stimulating hormone activity. 4. The apparent molecular weight of cFSH was 38,000 Da and a single band on isoelectric focusing had a pI of 4.65.     

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.     

Distribution and morphology of ghrelin immunostained cells in the adrenal gland of the African ostrich

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor. We investigated the distribution and morphological characteristics of ghrelin-immunopositive (ghrelin-ip) cells in the African ostrich adrenal gland. We found that the adrenal gland of the African ostrich consisted of three parts: capsule, inter-renal tissue and chromaffin cells. The inter-renal tissue and chromaffin cells interdigitated irregularly. The inter-renal tissue consisted of a peripheral zone and a central inner zone. The peripheral zone could be divided into an outer subcapsular zone and an inner zone. The subcapsular zone cells were arranged as a bow, while the inner area cells formed cords that were perpendicular to the capsule. The central inner zone exhibited irregular clumps and the cells were morphologically similar to chromaffin cells. Ghrelin-ip cells were located throughout the adrenal gland except the capsule. The majority of ghrelin-ip cells were found among the chromaffin cells. The number of ghrelin-ip cells in the inter-renal tissue decreased gradually from the central inner zone, to the inner zone to the subcapsular zone. The ghrelin-ip cells were oval or irregular in shape and exhibited cytoplasmic staining. Our findings suggest that ghrelin may play a role in regulating adrenal hormone secretion in the African ostrich.     

Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in the cerebellum.     

Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus)

Gonadotropin-releasing hormone (GnRH) molecular forms were studied in extracts of ostrich hypothalamus and extrahypothalamic brain using high performance liquid chromatography, radioimmunoassay with region-specific antisera and assessment of luteinizing hormone (LH)-releasing activity using chicken dispersed pituitary cells. Two molecular forms of GnRH with chromatographic, immunological and biological properties identical to those of Gln8-GnRH and His5,Trp7,Tyr8-GnRH were demonstrated in both the hypothalamic and extrahypothalamic brain extracts. A greater proportion of His5,Trp7,Tyr8-GnRH was present in the hypothalamus than in extrahypothalamic brain. It is likely that these two forms of GnRH are present in all bird species, since the chicken and the ostrich have evolved separately.     

Mesotocin and vasotocin, two neurohypophysial hormones in the ostrich, Struthio camelus

Two neurohypophysial hormones have been isolated from an avian species, the ostrich, Struthio camelus. Both have been characterized by amino acid analysis and sequence determination. The data obtained suggest that the oxytocin-like hormone is [Ile8-oxytocin] (mesotocin) and the vasopressin-like hormone is [Ile3-vasopressin] (vasotocin). Bioactivity measurements based on urinary conductivity showed vasotocin to be about five times as active as mesotocin.