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.

A COMPARISON OF PREPARATIONS OF HIGHLY PURIFIED HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE: DIFFERENCES IN THE FOLLICLE-STIMULATING HORMONE POTENCIES AS DETERMINED BY IN-VIVO BIOASSAY, IN-VITRO BIOASSAY AND IMMUNOASSAY

1981 ◽  
Vol 91 (2) ◽  
pp. 353-362 ◽  
Author(s):  
P. L. STORRING ◽  
A. A. ZAIDI ◽  
Y. G. MISTRY ◽  
BERIT FRÖYSA ◽  
BRIDGET E. STENNING ◽  
...  
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
In Vitro Bioassay ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Potency ◽  
Reference Preparation ◽  
In Vivo Bioassay

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A1%1 cm 280 = 10. The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH. Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay. The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals Evidence for N-linked glycosylation in Toxoplasma gondii

1993 ◽  
Vol 291 (3) ◽  
pp. 713-721 ◽  
Author(s):  
M Odenthal-Schnittler ◽  
S Tomavo ◽  
D Becker ◽  
J F Dubremetz ◽  
R T Schwarz
Keyword(s):  
Toxoplasma Gondii ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Common Precursor ◽  
The Common ◽  
Oligosaccharide Structures

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

PHYSIOLOGICAL ACTIONS OF HUMAN FOLLICLE-STIMULATING HORMONE AND ITS β-SUBUNIT IN REPTILES

1977 ◽  
Vol 74 (3) ◽  
pp. 441-447 ◽  
Author(s):  
PAUL LICHT ◽  
ANTONELLA BONA GALLO ◽  
ANNE STOCKELL HARTREE ◽  
RATNA C. SHOWNKEEN
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
Β Subunit ◽  
Binding Assays ◽  
Mammalian Tissues ◽  
Stimulation Of ◽  
Stimulating Hormone

SUMMARY The actions of human follicle-stimulating hormone (hFSH) and its β-subunit were examined in several assays in reptiles, including effects on lizard testicular activity (growth and androgen production) in vivo, and stimulation of androgen production by snake testes and competition for binding of 125I-labelled hFSH in lizards and snakes in vitro. Binding was also examined with mammalian tissues. The hFSH was highly steroidogenic in the snake and lizard; otherwise results were similar to those observed in mammals. In all cases, the potency of the β-subunit was only a few per cent of the intact hormone. The potency of hFSH in vivo compared with NIH-FSH ovine standards was several 100 times greater than in vitro. Results for stimulation of androgen production in vivo closely paralleled those for binding assays in both reptiles and mammals. In contrast to previous results for ovine FSH β-subunit, human FSH β-subunit has little if any FSH biological activity in reptiles.

scholarly journals The Multifunctional Bacteriophage P2 Cox Protein Requires Oligomerization for Biological Activity

2000 ◽  
Vol 182 (23) ◽  
pp. 6714-6723 ◽  
Author(s):  
Jesper M. Eriksson ◽  
Elisabeth Haggård-Ljungquist
Keyword(s):  
Biological Activity ◽  
Gel Filtration ◽  
Native Form ◽  
Host Chromosome ◽  
Multifunctional Protein ◽  
Bacteriophage P2 ◽  
Vitro Analysis ◽  
In Vitro Analysis

ABSTRACT The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 PLL promoter that controls P4 DNA replication. In this case it binds upstream of the PLL promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage λ cI-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing differentcox mutants and that oligomerization is mediated by the C-terminal region.

scholarly journals Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 954-960 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Binding Affinity ◽  
Half Life ◽  
Chinese Hamster ◽  
Β Subunit ◽  
Wild Type ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

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

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.

scholarly journals Studies on rat ovarian receptors for lutropin (luteinizing hormone). Interaction with β-subunit of sheep lutropin

1976 ◽  
Vol 160 (3) ◽  
pp. 615-619 ◽  
Author(s):  
K Muralidhar ◽  
N R Moudgal
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Chorionic Gonadotropin ◽  
Lower Order ◽  
Ovarian Response ◽  
Β Subunit ◽  
Α Subunit ◽  
Human Choriogonadotropin

By using radioimmunoassay, the interaction of sheep lutropin (luteinizing hormone, LH) β-subunit with rat ovarian receptors was investigated. The binding of β-subunit was specific, although of much lower order than that of lutropin. Sheep lutropin β-subunit effectively inhibited the binding of human choriogonadotropin (chorionic gonadotropin, gCG) to the ovary, showing that both occupy the same sites. The binding of sheep lutropin β-subunit to ovary was not followed by any detectable increase in cyclic AMP. The ovarian response to lutropin in terms of cyclic AMP production was inhibited in the presence of free β-subunit. The α-subunit of lutropin, when used at concentrations where contamination with whole lutropin was negligible, enhanced the degree of binding of β-subunit; this did not lead to increased cyclic AMP in the tissue. Surprisingly, the binding of β-subunit in vitro was drastically decreased by the prior removal of all endogenous rat lutropin bound to receptors. The implications of these data are discussed in the light of the reported biological activity of the β-subunit.

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

1980 ◽  
Vol 58 (4) ◽  
pp. 272-280 ◽  
Author(s):  
A. Fournier ◽  
R. Couture ◽  
J. Magnan ◽  
M. Gendreau ◽  
D. Regoli ◽  
...  
Keyword(s):  
Substance P ◽  
Solid Phase ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Phase Method ◽  
Elemental Analyses ◽  
Solid Phase Method

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure–activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.

Sign in / Sign up

Export Citation Format

Share Document