Isolation of FSH from bovine pituitary glands

1993 ◽  
Vol 137 (1) ◽  
pp. 59-68 ◽  
Author(s):  
J.-B. Wu ◽  
P. G. Stanton ◽  
D. M. Robertson ◽  
M. T. W. Hearn
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
High Yield ◽  
Purification Procedure ◽  
In Vitro Bioassay ◽  
Α Subunit ◽  
Pituitary Glands

ABSTRACT An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5·7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay. SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19·5 kDa and 15·8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with α- and β-subunits, and a third minor (< 20%) sequence consistent with the α-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators. Journal of Endocrinology (1993) 137, 59–68

Nucleotide (cDNA) sequence encoding the horse gonadotrophin α-subunit

1987 ◽  
Vol 115 (2) ◽  
pp. 341-346 ◽  
Author(s):  
F. Stewart ◽  
J. A. Thomson ◽  
S. E. A. Leigh ◽  
J. M. Warwick
Keyword(s):  
Amino Acid ◽  
Peptide Sequencing ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Northern Hybridization ◽  
Cdna Clones ◽  
Subunit Gene ◽  
Α Subunit ◽  
Pituitary Glands

ABSTRACT Several cDNA clones corresponding to mRNA for the α-subunit of the horse (Equus caballus) pituitary and placental (chorionic) gonadotrophic hormones have been isolated and sequenced. Polyadenylated mRNA was purified from horse pituitary glands (the source of FSH and LH) and horse placental tissues (the source of chorionic gonadotrophin; CG). The mRNA preparations were characterized by in-vitro translation and Northern hybridization techniques using human and ovine gonadotrophin cDNA clones as probes. Complementary DNA libraries were created from the pituitary and placental mRNAs and a human CG α-subunit probe was used to isolate several horse α-subunit cDNA clones. The α-subunit nucleotide sequence from both sources of tissue was identical, thereby indicating that in the horse (as in man) the same gonadotrophin α-subunit gene is expressed in the pituitary and placenta. Our results are consistent with transcription of a single α-subunit gene for all the glycoprotein hormones in the horse, and we suggest that the reported differences between the horse CG and FSH α-subunit amino acid sequences determined by conventional peptide sequencing methods arose due to errors in the FSH α-subunit sequence. Comparison of the deduced amino acid sequence of the horse α-subunit with that of other α-subunit sequences indicated a number of significant differences which may be related to the unusual receptor-binding properties of the equine gonadotrophins. J. Endocr. (1987) 115, 341–346

Isolation of Somatostatin (a Somatotropin Release Inhibiting Factor) of Ovine Hypothalamic Origin

1974 ◽  
Vol 52 (11) ◽  
pp. 1067-1072 ◽  
Author(s):  
P. Brazeau ◽  
W. Vale ◽  
R. Burgus ◽  
R. Guillemin
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partition Chromatography ◽  
Inhibiting Factor ◽  
Layer Chromatography

Isolation of somatostatin, a tetradecapeptide of ovine origin inhibiting somatotropin secretion, is reported. About 490 000 hypothalamic fragments were submitted to alcohol–chloroform extraction, countercurrent distribution, ion-exchange chromatography, gel filtration, and partition chromatography. Of the 8.5 mg material thus obtained, 77% was accounted for by a peptide shown homogeneous by electrophoresis, thin-layer chromatography, and amino acid analysis. The peptide inhibits the secretion of radioimmunoassayable growth hormone at doses of ≥ 1.0 nM in vitro and 400 ng per rate in vivo.

Isolation and Partial Characterization of Adipotropin, an Apparent New Lipolytic Peptide from Bovine Anterior Pituitary Lobes

1980 ◽  
Vol 35 (9) ◽  
pp. 1171-1177 ◽  
Author(s):  
Karen O’Malley ◽  
Karl Folkers ◽  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Anterior Pituitary ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Frozen Tissue ◽  
Pituitary Glands ◽  
Approximate Molecular Weight ◽  
One Year

A peptide, apparently new, has been isolated from the anterior lobes of bovine pituitary glands by the following steps: (1) lyophilization of frozen tissue; (2) homogenization; (3) extraction with an aqueous buffer; (4) gel filtration; (5) anion and then (6) cation exchange chromatography; (7) HPLC. One antioxidant and two proteolytic inhibitors were present during buffer extraction to avoid artifactual reactions. Amino acid analyses consistently revealed the same amino acids and no others on different specimens, one year apart. The composition was estimated as: 4 Asp, 2 Thr, 5 Ser, 8 Glu(x), 5 Pro, 10 Gly, 4 Ala, 2 Val, 2 Met, 1 Ile, 2 Leu, 1 Tyr, 1 Phe, 2 His, 3 Lys, 2 Arg (5600 daltons). Cysteine and tryptophan were absent. An approximate molecular weight by gel filtration was 7200 daltons. The isoelectric point was 6.1. The peptide showed a dose-response activity in the range of 0.001-1.0 μg in a rabbit adipose system, in vitro. The peptide is tentatively designated as adipotropin, because it has no unequivocal chemical relation­ship to other relevant lipolytic peptides, and because it has the highest molar potency, in vitro, of these peptides, including pACTH.

A NEUROPHYSIN FROM COD (GADUS MORRHUA) PITUITARY GLANDS: ISOLATION AND PROPERTIES

1968 ◽  
Vol 42 (1) ◽  
pp. 143-NP ◽  
Author(s):  
B. T. PICKERING
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Composition ◽  
Arginine Vasopressin ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Maximum Binding Capacity ◽  
Pituitary Glands

SUMMARY A protein capable of binding neurohypophysial hormones has been isolated from cod pituitary glands using gel filtration and ion-exchange chromatography. The cod protein which was acidic and rich in cystine had an amino acid composition closely related to those of the mammalian neurophysins. It had a maximum binding capacity of 2·2 μmole/14mg. for oxytocin, 2·1 μmole/14 mg. for [8-arginine]-oxytocin and 1·1 μmole/14 mg. for [8-arginine]-vasopressin. Thus the cod protein had a greater capacity for the endogenous pressor-antidiuretic peptide than for the analogous mammalian hormone.

PURIFICATION AND PROPERTIES OF THE SUBUNITS OF HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE

1976 ◽  
Vol 69 (2) ◽  
pp. 263-273 ◽  
Author(s):  
RATNA C. SHOWNKEEN ◽  
ANNE STOCKELL HARTREE ◽  
FRANCESCA STEWART ◽  
K. MASHITER ◽  
V. C. STEVENS
Keyword(s):  
Biological Activity ◽  
Sialic Acid ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Β Subunit ◽  
Human Pituitary ◽  
High Biological Activity ◽  
Α Subunit

SUMMARY Highly purified human pituitary FSH was partially dissociated by treatment with 8 m-urea, and α- and β-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated α-subunit had less than 1%, and β-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with the purified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the β-subunit.

Bovine Factors X1And X2: Activation Peptide Based Chromatographic Differences

1979 ◽  
Author(s):  
Takashi Morita ◽  
Craig Jackson
Keyword(s):  
Amino Acid ◽  
Anion Exchange ◽  
Gel Filtration ◽  
Heart Association ◽  
Personal Communication ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Factor X ◽  
Activation Peptide ◽  
Activation Peptides

Bovine Factor X is eluted in two forms (X1and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglutamic acid residues have been shown to be identical in X1 and X2(H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by anion-exchange chromatography. The activation peptide (AP1) which eluted first was shown to be derived from Factor X1. while the activation peptiae (AP2) which eluted second was shown to be derived from X2 on the basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.M.J. is an Established Investigator of the American Heart Association).

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

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