Receptors for lactogenic hormones in the ovine corpus luteum. III: Inhibition of 125I-labelled human growth hormone binding by a high molecular weight factor in ovine corpus luteum cytosol

1987 ◽  
Vol 114 (3) ◽  
pp. 383-389 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies ◽  
A. S. McNeilly ◽  
H. G. Friesen
Keyword(s):  
Molecular Weight ◽  
Corpus Luteum ◽  
Metal Ion ◽  
Specific Binding ◽  
Human Growth ◽  
Gel Chromatography ◽  
Hormone Binding ◽  
Cytosol Fraction ◽  
Dose Dependent Fashion ◽  
Ovine Prolactin

ABSTRACT Ovine luteal cytosol fractions inhibited the specific binding of 125I-labelled human GH and ovine prolactin (oPRL) to ovine luteal microsomes in a dose-dependent fashion. Inhibition was dependent on divalent cation concentrations, and was abolished by divalent metal ion chelating agents or by boiling. Inhibition was not due to ionic strength or salt effects on hormone binding, the release of endogenously bound oPRL into the cytosol fraction during tissue disruption and fractionation, or the presence of a soluble (or solubilized) lactogenic receptor in ovine cytosol preparations. Gel chromatography of cytosol fractions gave a molecular weight for the inhibitor of approximately 50 000. J. Endocr. (1987) 114, 383–389

Receptors for lactogenic hormones in the ovine corpus luteum. I: A major discrepancy in the specific binding of radiolabelled ovine prolactin and human growth hormone

1987 ◽  
Vol 113 (3) ◽  
pp. 365-374 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies ◽  
A. S. McNeilly ◽  
H. G. Friesen
Keyword(s):  
Cell Surface ◽  
Corpus Luteum ◽  
Specific Binding ◽  
Surface Membrane ◽  
Human Growth ◽  
Luteal Cell ◽  
Cell Surface Membrane ◽  
Luteal Tissue ◽  
Lactogenic Hormones ◽  
Ovine Prolactin

ABSTRACT The characteristics of the binding of 125I-labelled human GH (hGH) and ovine prolactin (oPRL) were studied in the ovine corpus luteum. Although oPRL is the homologous ligand for sheep lactogenic receptors, its binding was significantly and consistently lower than that of 125I-labelled hGH. This was not due to iodination damage of oPRL since: (1) 125I-labelled oPRL tracers which bound poorly relative to 125I-labelled hGH in the ovine corpus luteum were equipotent in the pig and rat corpus luteum, (2) the differences between 125I-labelled hGH and oPRL binding persisted with tracers of equivalent biopotency and (3) the iodination procedure affected neither oPRL bioactivity in the Nb2 tumour assay nor its binding activity with ovine corpus luteum receptors. Ovine luteal receptors were specific for lactogenic hormones. The specific binding of 125I-labelled hGH or oPRL could be inhibited completely by incubation with either unlabelled hormone, with similar potencies. However, oGH inhibited binding only at much higher concentrations, consistent with its known contamination with oPRL. Moreover, 125I-labelled oGH was not bound specifically to sheep luteal tissue. Fractionation of sheep luteal homogenates on sucrose density gradients (with or without cell-surface membrane perturbation by digitonin) demonstrated that binding of 125I-labelled hGH and 125I-labelled oPRL peaked in the same regions of the gradients, coincident with a number of luteal cell-surface membrane markers. We conclude that the marked discrepancy between the binding of hGH and oPRL tracers by sheep luteal tissue was not due to iodination damage of oPRL, binding of 125I-labelled hGH to somatogenic receptors or differential binding to luteal cell-surface versus intracellular receptors. J. Endocr. (1987) 113, 365–374

Specific binding sites for ovine prolactin in three amphibian cell lines

1985 ◽  
Vol 248 (1) ◽  
pp. C80-C87 ◽  
Author(s):  
M. Dunand ◽  
M. L. Aubert ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier
Keyword(s):  
Growth Hormone ◽  
Cell Lines ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Growth ◽  
Functional Differentiation ◽  
Water Metabolism ◽  
Ovine Prolactin ◽  
Follicle Stimulating Hormones

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.

PROLACTIN RECEPTORS IN THE MAMMARY GLAND, CORPUS LUTEUM AND OTHER TISSUES OF THE TAMMAR WALLABY, MACROPUS EUGENII

1979 ◽  
Vol 83 (1) ◽  
pp. 79-89 ◽  
Author(s):  
C. SERNIA ◽  
C. H. TYNDALE-BISCOE
Keyword(s):  
Mammary Gland ◽  
Corpus Luteum ◽  
Specific Binding ◽  
Tammar Wallaby ◽  
Binding Activity ◽  
Breeding Cycle ◽  
Macropus Eugenii ◽  
Lactating Mammary Gland ◽  
The Mean ◽  
Ovine Prolactin

SUMMARY Specific binding of radio-iodinated ovine prolactin to subcellular tissue fractions of the tammar wallaby (Macropus eugenii) was investigated. Specific binding was found, in order of decreasing binding activity, in the lactating mammary gland, corpus luteum, corpus albicans, adrenal gland and ovary. Specific binding was absent in kidney, liver, brain and inactive mammary gland. The mean association constant (Ka at 23 °C) was determined as 0·90 × 109, 2·20 × 109, 2·44 × 109, 3·38 × 109 and 10·98 × 1091/mol for mammary gland, adrenal, corpus albicans, corpus luteum and ovary respectively. The mean receptor concentration (N) varied from 92·87 × 10−14 mol/mg protein for the mammary gland to 1·03 × 10−14 mol/mg protein for the ovary. The concentration in the corpus luteum varied between tissue pools collected at different times of the annual breeding cycle. The specificity for prolactin was shown in the mammary gland and corpus luteum by the failure of ovine FSH, LH, GH and TSH to displace 125I-labelled ovine prolactin, whereas it was displaced readily by both ovine and bovine prolactin.

Receptors for lactogenic hormones in the ovine corpus luteum. II: Specific inactivation of ovine prolactin

1987 ◽  
Vol 113 (3) ◽  
pp. 375-381 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies ◽  
A. S. McNeilly ◽  
H. G. Friesen
Keyword(s):  
Corpus Luteum ◽  
Protease Inhibitors ◽  
Binding Activity ◽  
Gel Chromatography ◽  
Elution Profile ◽  
Binding Ability ◽  
Receptor Binding Activity ◽  
Luteal Tissue ◽  
Lactogenic Hormones ◽  
Ovine Prolactin

ABSTRACT Sheep corpus luteum homogenates and membrane fractions discriminate between 125I-labelled human GH (hGH) and ovine prolactin (oPRL). The present studies were designed to establish whether ovine luteal tissue possessed a prolactin-specific inactivating enzyme. Preincubation of sheep luteal microsomes and cytosol fractions with 125I-labelled hGH had little effect on the ability of the hormone to rebind to pig luteal lactogenic receptors. In contrast, sheep luteal tissue fractions markedly decreased the binding ability of 125I-labelled oPRL. However, despite the profound loss of receptor-binding activity, there was no change in protein-bound radioactivity, nor in the elution profile of 125I-labelled oPRL by gel chromatography on Sephadex G-100. Moreover, the disparity between 125I-labelled hGH and oPRL was not overcome by preincubation of sheep luteal membranes with protease inhibitors of differing specificities. We conclude that the disparity between the binding of hGH and oPRL in ovine tissues was due to the selective inactivation of oPRL. However, the activity responsible did not degrade the hormone extensively, nor was its action blocked by a range of protease inhibitors. J. Endocr. (1987) 113, 375–381

Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

1993 ◽  
Vol 70 (04) ◽  
pp. 625-630 ◽  
Author(s):  
Edward Young ◽  
Benilde Cosmi ◽  
Jeffrey Weitz ◽  
Jack Hirsh
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Plasma Proteins ◽  
Low Molecular Weight Heparin ◽  
Specific Binding ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

Identification of Fibrinogen Derivatives in Plasma Samples

1972 ◽  
Vol 27 (03) ◽  
pp. 610-618 ◽  
Author(s):  
H Graeff ◽  
R von Hugo
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Plasma Samples ◽  
Methodological Study ◽  
Parent Molecule ◽  
Electrophoretic Mobilities ◽  
Fibrin Monomer

SummaryThe observation of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of DIC initiated the present methodological study. These derivatives were identified by the following methods : 2.5 M β-alanine precipitation of the plasma samples, PAA gel electrophoresis, intra gel immunoprecipitation and agarose gel chromatography. In the plasma of a patient with severe eclampsia and laboratory signs of DIC two derivatives with a molecular weight higher than that of fibrinogen were identified according to their relative electrophoretic mobilities: 0.18 and 0.28 × 10−5 cm2/V × sec (fibrinogen: 0.43 × 10−5 cm2/V × sec). Electrophoretic studies in the presence of 5 M urea indicated that the 0.28 derivative is a complex probably formed by fibrinogen and a fibrin monomer.

The Relation Between VIIIR:AG and VIII:C Studied with Two Antisera

1979 ◽  
Author(s):  
H. P. Muller ◽  
N. H. van Tilburg ◽  
R. M. Bertina ◽  
J. J. Veltkamp
Keyword(s):  
Molecular Weight ◽  
High Salt ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Specific Antibodies ◽  
Specific Effects ◽  
Normal Plasma ◽  
Coagulant Activity ◽  
Sterical Hindrance ◽  
Inhibitory Antibodies

FVIII was separated into low molecular weight FVIII (LMW FVIII) and high molecular weight FVIII (HMW FVIII) by gel chromatography in the presence of high salt concentration or by high salt elution of LMW FVIII from FVIII bound to anti HMW FVII-Sepharose. Specific antibodies were raised in rabbits against HMW FVIII and LMW FVIII. After removal of the contaminating anti HMW activities the rabbit anti LMW FVIII was still able to neutralize the FVIII coagulant activity of normal plasma and of IMW FVIII with canparable efficiency and it had no effect on the VIIIR:WF of FVIII in normal plasma or in HMW FVIII. Anti LMW FVIII does not bind to HMW FVIII and does not precipitate FVIII as tested by counter immunoelectrophoresis. Rabbit anti HMW FVIII precipitates FVIII in normal plasma, inhibits VIIIR:WF activity, while it has no effect on the FVIII coagulant activity of LMW FVIII. The coagulant activity of FVIII in normal plasma is slightly inhibited by anti HMW FVIII presumably by non-specific effects (sterical hindrance). It is concluded that inhibitory antibodies against VIII:C raised in rabbits recognize antigenic structures only present on LMW FVIII. Antibodies against HMW FVIII raised in rabbits appears to recognize structures only present on HMW FVIII.

Polysaccharides from the Flowers of Malva silvestris L., ssp. mauritiana (L.) THELL.: The Structure of D-Glucan

1992 ◽  
Vol 57 (11) ◽  
pp. 2400-2406 ◽  
Author(s):  
Peter Capek
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Average Molecular Weight ◽  
Linear Structure ◽  
Gel Chromatography ◽  
Spectroscopic Investigations ◽  
Glycosidic Bonds

The neutral polysaccharide α-D-glucan was isolated from the flowers of Malva silvestris L., ssp. mauritiana (L.) THELL. using a combination of ion exchange and gel chromatography. It was homogeneous under the conditions of free electrophoresis of average molecular weight Mn 25260. The chemical and spectroscopic investigations indicated a linear structure of the polysaccharide in which the α-D-glucopyranose units were linked predominantly by 1→6 glycosidic bonds, while some saccharides were the place of branching in position C-3.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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