scholarly journals Binding and structural characteristics of a soluble lactogen-binding protein from rabbit mammary-gland cytosol

1986 ◽  
Vol 237 (3) ◽  
pp. 813-820 ◽  
Author(s):  
S I Ymer ◽  
A C Herington
Keyword(s):  
Mammary Gland ◽  
High Speed ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Binding Protein ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Scatchard Analysis ◽  
Specific Binding Protein

Specific receptors for prolactin (PRL) are known to be present on plasma membranes and intracellular membranes of mammary gland. We now report, however, the detection and characterization of a soluble lactogen-specific binding protein in high-speed (200,000 g) cytosolic preparations from pregnant- and non-pregnant-rabbit mammary gland. The binding protein was not detectable by poly(ethylene glycol) precipitation; instead, bound and free 125I-labelled human growth hormone (hGH; a potent lactogen) was separated using a mini-gel filtration technique. Specific binding of 125I-hGH reached an apparent equilibrium between 10 and 14 h at 21-23 degrees C. It was dependent on mammary-gland protein concentration and, partially, on Ca2+ or Mg2+ concentrations. Scatchard analysis revealed steep curvilinear plots, the high-affinity component having a KA of approximately 3 × 10(10) M-1. Gel filtration on calibrated Ultrogel AcA34 columns of 125I-hGH-cytosol complexes or of cytosol alone, followed by measurement of 125I-hGH binding in each eluted fraction, indicated that the binding protein had an Mr of 33,000-43,000. A specific binding protein of the same size was observed when 125I-ovine or -human PRL, but not 125I-bovine GH, was used as ligand. The apparent lactogenic specificity was confirmed by a lack of cross-reactivity of the binding protein with an anti-[GH receptor (rabbit liver)] monoclonal antibody. Polyacrylamide-gel electrophoresis of 125I-hGH covalently cross-linked to cytosol with disuccinimidyl suberate revealed binding proteins of Mr 35,000 (non-reduced) and 37,000 (reduced), results comparable with those obtained by gel filtration and indicating an absence of inter-subunit disulphide bonds. These studies have shown the presence of an apparently naturally soluble lactogen-binding protein in the cytosolic fraction of rabbit mammary gland. The relationship between this binding protein and the membrane PRL receptor is not yet known.

Endonexin II, Present on Human Liver Plasma Membranes, Is a Specific Binding Protein of Small Hepatitis B Virus (HBV) Envelope Protein

Virology ◽  
1993 ◽  
Vol 197 (2) ◽  
pp. 549-557 ◽  
Author(s):  
Kurt Hertogs ◽  
William P.J. Leenders ◽  
Erick Depla ◽  
Wieke C.C. De Bruin ◽  
Lydie Meheus ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Human Liver ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Binding Protein ◽  
Liver Plasma Membranes ◽  
B Virus ◽  
Specific Binding Protein

Cyclic adenosine 3′,5′-monophosphate binding protein in developing myxospores of Myxococcus xanthus

1980 ◽  
Vol 26 (8) ◽  
pp. 905-911 ◽  
Author(s):  
Michael Orlowski
Keyword(s):  
Binding Sites ◽  
High Speed ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Myxococcus Xanthus ◽  
Protein Band ◽  
Specific Protein ◽  
Supernatant Fraction ◽  
Scatchard Analysis ◽  
Single Class

The interaction of cyclic adenosine 3′,5′-monophosphate (cAMP) with specific protein molecules was examined in the high-speed supernatant fraction of extracts made at stages throughout glycerol-induced myxospore development in Myxococcus xanthus. Experiments using 8-azido[32P]cAMP, a photoaffinity analogue of cAMP, and SDS – polyacrylamide gel electrophoresis showed that the nucleotide interacts with only a single protein band of 12 500 molecular weight. Both the identiy and amount of this protein remained constant throughout development. The binding protein was specific for cAMP; other nucleotides did not compete with cAMP for binding sites. A Scatchard analysis showed evidence of only a single class of binding sites with a high affinity for cAMP.

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

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
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.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

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