SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

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Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)55434-x ◽

1990 ◽

Vol 265 (26) ◽

pp. 15564-15571 ◽

Cited By ~ 9

Author(s):

J. Vázquez ◽

P. Feigenbaum ◽

V.F. King ◽

G.J. Kaczorowski ◽

M.L. Garcia

Keyword(s):

Rat Brain ◽

Binding Sites ◽

Plasma Membranes ◽

Affinity Binding ◽

Synaptic Plasma Membranes ◽

High Affinity Binding ◽

High Affinity ◽

Direct Association ◽

Voltage Dependent

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Fungal Peptide Elicitors: Signals Mediating Pathogen Recognition in Plants

Zeitschrift für Naturforschung C ◽

10.1515/znc-1998-3-401 ◽

1998 ◽

Vol 53 (3-4) ◽

pp. 141-150 ◽

Cited By ~ 8

Author(s):

Thorsten Nürnberger ◽

Dirk Nennstiel

Keyword(s):

Plant Defense ◽

Binding Sites ◽

Plasma Membranes ◽

Pathogen Resistance ◽

Microbial Pathogens ◽

Functional Link ◽

High Affinity ◽

Highly Sensitive ◽

Recognition Systems

Abstract Highly sensitive and specific recognition systems for microbial pathogens are essential for disease resistance in plants. Proteinaceous elicitors activating plant pathogen defense have been identified in numerous antagonistic plant/fungus interactions. Precisely defined signal structures required for elicitor-mediated activation of plant defense are indicative of the involvement of receptors in elicitor perception and subsequent signal generation. Use of pure elicitor preparations has helped to establish a functional link between binding of elicitors to high-affinity binding sites in plant plasma membranes and activation of plant defense. Thus elicitor binding sites appear to function as physiological receptors. Currently, isolation and molecular characterization of elicitor receptors is under way. Transfer of new recognition specificities into plants is supposed to be a key strategy for engineering pathogen resistance in economically important crops.

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New aspects of the physiological significance of LRH receptors of pituitary plasma membranes

Acta Endocrinologica ◽

10.1530/acta.0.0960036 ◽

1981 ◽

Vol 96 (1) ◽

pp. 36-45 ◽

Cited By ~ 4

Author(s):

Herbert Kuhl ◽

Rudolf Baumann

Keyword(s):

Binding Sites ◽

Plasma Membranes ◽

Functional Relationship ◽

Metabolic Pattern ◽

High Affinity ◽

Highly Active ◽

Rat Pituitary ◽

Intracellular Compartments ◽

Triton X ◽

Two Peaks

Abstract. It has been found that l-cystine-bis-(4-nitroanilide), a synthetic peptidase substrate, competes with [125I]LRH for specific high affinity LRH receptors on isolated rat pituitary plasma membranes as effectively as a highly active LRH analogue. The pattern of [125I]LRH degradation by isolated pituitary plasma membranes was similar to that effected by pituitary and hypothalamic enzyme extracts. The enzyme activity seemed to accumulate in the supernatant of the membrane suspension after being released from the membrane structure. Plasma membrane supernatant and pituitary and hypothalamic enzyme extracts were chromatographed together with labelled LRH on Sephadex G 200. This resulted in protein binding patterns with one peak at the fractions representing a molecular weight of 600 000 to 650 000 and another one of approximately 80 000, the first probably being an aggregate of the latter. After solubilization of the plasma membranes by Triton X-100, there was no evidence for another LRH receptor. There were also two peaks of [125I]LRH degrading activity (with identical metabolic pattern) and two peaks of arylamidase activity, which were, however, not identical with the binding peaks. Both the arylamidase and the LRH binding-pattern had been demonstrated to be temperature- and hormonedependent, and seemed to be functionally related. The results indicate that there is a functional relationship between LRH binding-sites on plasma membranes and LRH degrading enzymes. This implies that the search for the 'true' LRH receptor has to be directed towards intracellular compartments of the pituitary gonadotrophs.

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Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657167 ◽

1982 ◽

Vol 47 (03) ◽

pp. 197-202 ◽

Cited By ~ 23

Author(s):

Kurt Huber ◽

Johannes Kirchheimer ◽

Bernd R Binder

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Human Urine ◽

Gel Filtration ◽

Chain Structure ◽

The Other ◽

Single Chain ◽

Apparent Homogeneity ◽

Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)33206-4 ◽

1976 ◽

Vol 251 (16) ◽

pp. 4947-4957

Author(s):

V K Bhalla ◽

J Haskell ◽

H Grier ◽

V B Mahesh

Keyword(s):

Binding Sites ◽

Partial Characterization ◽

Rat Testis ◽

High Affinity ◽

Binding Factor

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Characterization of the binding of plasminogen activators to plasma membranes from human liver

Biochemical Journal ◽

10.1042/bj2870911 ◽

1992 ◽

Vol 287 (3) ◽

pp. 911-915 ◽

Cited By ~ 5

Author(s):

G Nguyen ◽

S J Self ◽

C Camani ◽

E K O Kruithof

Keyword(s):

Human Liver ◽

Plasma Membranes ◽

Gel Filtration ◽

Mannose Receptor ◽

Hepatoma Cells ◽

Tissue Type ◽

High Affinity ◽

Liver Membranes ◽

Membrane Bound ◽

Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

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Characterization of high affinity binding sites for vasopressin in bovine adrenal medulla

Neuropeptides ◽

10.1016/0143-4179(84)90116-1 ◽

1984 ◽

Vol 4 (5) ◽

pp. 413-420 ◽

Cited By ~ 15

Author(s):

Ferenc A. Antoni

Keyword(s):

Adrenal Medulla ◽

Binding Sites ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Bovine Adrenal Medulla

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Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

Biochemical Journal ◽

10.1042/bj2410063 ◽

1987 ◽

Vol 241 (1) ◽

pp. 63-70 ◽

Cited By ~ 8

Author(s):

Y Ikehara ◽

Y Hayashi ◽

S Ogata ◽

A Miki ◽

T Kominami

Keyword(s):

Alkaline Phosphatase ◽

Phospholipase C ◽

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Soluble Form ◽

Microsomal Membranes ◽

Hydrophobic Nature ◽

Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

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