Properties of soluble somatostatin-binding protein

A soluble somatostatin-binding protein was detected in the cytosol fractions of various rat, human and bovine tissues. Maximum binding occurred at pH8.0–8.5 and was Ca2+-dependent. The specific binding of somatostatin per 10μg of cytosol protein from 12 rat tissues ranged between 36 and 15%, and 3% for peripheral blood cells. There was also substantial binding in cytosol from human anterior pituitary and liver, and bovine anterior pituitary. The specific binding in rat and human plasma in the presence of EDTA was only 1%. Gel filtration suggested a molecular weight of approx. 80000 for the somatostatin-binding protein from several sources. Exposure of the binding protein to trypsin eliminates somatostatin-binding activity but ribonuclease and deoxyribonuclease have no effect. The binding protein is thermolabile, ethanol-precipitable, and not completely specific for somatostatin. Bound125I-labelled [Tyr1]somatostatin is not easily displaced by excess of unlabelled somatostatin. The effects of dithiothreitol and mercaptoethanol on the binding of125I-labelled [Tyr1]somatostatin to the binding protein suggests that binding involves two sequential steps, first loose binding, then disulphide linkage. Since semipurified somatostatin-binding protein causes a dose-related inhibition of the binding of125I-labelled [Tyr1]somatostatin in radioimmunoassays for somatostatin, estimates of somatostatin content of tissue extracts by radioimmunoassay in some cases may be spuriously high. It is not yet clear whether the binding protein is a true cytosol protein or an easily solubilized membrane protein.

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Characteristics of a somatostatin-binding protein

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y78-007 ◽

1978 ◽

Vol 56 (1) ◽

pp. 48-53 ◽

Cited By ~ 15

Author(s):

N. Ogawa ◽

T. Thompson ◽

H. G. Friesen

Keyword(s):

Diabetes Mellitus ◽

Binding Protein ◽

Gel Filtration ◽

The Other ◽

Rat Tissues ◽

Cytoplasmic Protein ◽

Disulfide Bridges ◽

Thiol Groups ◽

Tissue Extracts ◽

Streptozotocin Induced Diabetes

The concentrations of a somatostatin-binding protein, found in the cytosol of a number of rat tissues, are similar in both sexes, and hypophysectomy has little or no effect on the level of binding protein in tissue extracts. On the other hand, streptozotocin-induced diabetes mellitus causes a modest decrease. The somatostatin-binding proteins obtained from extracts of several rat tissues are not only similar in molecular weight but also exhibit a similar isoelectric point and electrophoretic mobility. Agents that block thiol groups or prevent the formation of disulfide bridges markedly decrease the binding of somatostatin to the cytoplasmic protein. Studies using thiol reagents and gel filtration suggest that free thiol groups in somatostatin-binding protein are important for the binding of somatostatin.

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A high-affinity oestrogen-binding protein in cockerel liver cytosol

Biochemical Journal ◽

10.1042/bj1800347 ◽

1979 ◽

Vol 180 (2) ◽

pp. 347-353 ◽

Cited By ~ 29

Author(s):

C B Lazier ◽

A J Haggarty

Keyword(s):

Binding Sites ◽

Proteinase Inhibitors ◽

Specific Binding ◽

Binding Protein ◽

Gel Filtration ◽

Binding Activity ◽

Single Class ◽

High Affinity ◽

Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

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Radioimmunoassay Studies of Intestinal Calcium-binding Protein in the Pig. II. The Distribution of Intestinal CaBP in Pig Tissues

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y75-158 ◽

1975 ◽

Vol 53 (6) ◽

pp. 1135-1140 ◽

Cited By ~ 27

Author(s):

B. M. Arnold ◽

M. Kuttner ◽

D. M. Willis ◽

A. J. W. Hitchman ◽

J. E. Harrison ◽

...

Keyword(s):

Small Intestine ◽

Calcium Binding ◽

Binding Protein ◽

Gel Filtration ◽

Protein S ◽

Binding Activity ◽

Calcium Binding Protein ◽

Distal Small Intestine ◽

Specific Radioimmunoassay ◽

Kidney Liver

Using a specific radioimmunoassay for porcine intestinal calcium-binding protein (CaBP), we have measured the concentration of CaBP in the various tissues and organs of normal pigs. Intestinal CaBP was present in highest concentration in the upper small intestine, with lower concentrations in the distal small intestine. Intestinal CaBP was also found, in lower concentrations, in kidney, liver, thyroid, pancreas, and blood. In all other tissues, including parathyroid, bone, skeletal muscle, and brain, CaBP immunoreactivity was undetectable or less than in blood. The elution profile of calcium-binding activity and immunoreactivity from gel filtration analysis of kidney and parathyroid extracts suggest that the calcium-binding protein in the parathyroid gland, and the major calcium-binding protein(s) in the kidney, are chemically and immunochemically different from intestinal CaBP.

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Fine mapping of Plasmodium falciparum ribosomal phosphoprotein PfP0 revealed sequences with highly specific binding activity to human red blood cells

Journal of Molecular Medicine ◽

10.1007/s00109-009-0533-5 ◽

2009 ◽

Vol 88 (1) ◽

pp. 61-74 ◽

Cited By ~ 3

Author(s):

Gabriela Arevalo-Pinzon ◽

Hernando Curtidor ◽

Claudia Reyes ◽

Martha Pinto ◽

Carolina Vizcaíno ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Fine Mapping ◽

Blood Cells ◽

Specific Binding ◽

Binding Activity ◽

Human Red Blood Cells

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Transcriptional and Posttranscriptional Regulation of Insulin-Like Growth Factor Binding Protein-3 by Cyclic Adenosine 3′,5′-Monophosphate: Messenger RNA Stabilization Is Accompanied by Decreased Binding of a 42-kDa Protein to a Uridine-Rich Domain in the 3′-Untranslated Region

Molecular Endocrinology ◽

10.1210/mend.13.3.0252 ◽

1999 ◽

Vol 13 (3) ◽

pp. 495-504

Author(s):

N. E. Erondu ◽

J. Nwankwo ◽

Y. Zhong ◽

M. Boes ◽

B. Dake ◽

...

Keyword(s):

Growth Factor ◽

Untranslated Region ◽

Specific Binding ◽

Binding Protein ◽

Binding Activity ◽

Insulin Like Growth Factor ◽

Factor Binding ◽

Mdbk Cells ◽

Camp Induction ◽

Igfbp 3

Abstract The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased ∼3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3′-untranslated region (3′-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

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Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity

Journal of Endocrinology ◽

10.1677/joe.0.1270139 ◽

1990 ◽

Vol 127 (1) ◽

pp. 139-148 ◽

Cited By ~ 21

Author(s):

C. Y. Lee ◽

D. M. Henricks

Keyword(s):

Growth Factor ◽

Bovine Serum ◽

Binding Proteins ◽

Binding Protein ◽

Gel Filtration ◽

Binding Activity ◽

Insulin Like Growth Factor ◽

Ethanol Extraction ◽

Factor I ◽

Igf I

ABSTRACT Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of Mr 150 000 and the other of Mr 40 000–45 000. The majority of the immunoreactive IGF-I was associated with the Mr 150 000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150 000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose–response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment. Journal of Endocrinology (1990) 127, 139–148

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THYROID HORMONE-BINDING INTERACTIONS IN CYTOSOL OF RAT ANTERIOR PITUITARY

Acta Endocrinologica ◽

10.1530/acta.0.0850256 ◽

1977 ◽

Vol 85 (2) ◽

pp. 256-266 ◽

Cited By ~ 3

Author(s):

Valerie Anne Galton

Keyword(s):

Thyroid Hormone ◽

Binding Sites ◽

Anterior Pituitary ◽

Binding Capacity ◽

Relative Effectiveness ◽

Binding Activity ◽

Rat Tissues ◽

Hormone Binding ◽

Cytosol Fraction ◽

Binding Interactions

ABSTRACT Thyroxine (T4) and triiodothyronine (T3)-binding interactions in preparations of rat anterior pituitary gland have been studied. T4 is bound primarily to extranuclear binding sites located in the cytosol fraction of the cell. These sites have a medium affinity for T4: Ka = 2.5 × 108 1/mol and a maximum binding capacity (MBC) of 1.15 pmol/mg tissue (wet weight). Binding of T3 to these sites is minimal. The extent of binding of T4 is influenced by the pH of the system and the temperature of incubation. The relative effectiveness of T4 analogues in displacing bound T4 is tetrac > T4 > triac > D-T4 > T3. Similar T4-binding sites are present in other rat tissues, but in all except serum, binding activity is lower than in the pituitary. T4-binding by serum contaminating the pituitary preparations contributed only partially to the total activity observed. Concomitant assessment of T4-binding activity and T4 metabolism in pituitary homogenates prepared at different pH values indicated an inverse relationship between the two processes. The possible role of thyroid hormone binding in cytosol in influencing the intracellular distribution of thyroid hormones is discussed.

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Solubilization and purification of a membrane-associated 3,3′,5-tri-iodo-l-thyronine-binding protein from rat erythrocytes

Biochemical Journal ◽

10.1042/bj2700577 ◽

1990 ◽

Vol 270 (3) ◽

pp. 577-582 ◽

Cited By ~ 3

Author(s):

R C Angel ◽

J A Botta ◽

R D Morero ◽

R N Farias

Keyword(s):

Hormone Receptors ◽

Specific Binding ◽

Binding Protein ◽

Active Form ◽

Binding Activity ◽

Erythrocyte Membranes ◽

Major Protein ◽

Sds Page ◽

Affinity Labelling ◽

Zwitterionic Detergent

3,3′,5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.

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ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation

Molecular and Cellular Biology ◽

10.1128/mcb.01598-07 ◽

2007 ◽

Vol 28 (2) ◽

pp. 705-717 ◽

Cited By ~ 154

Author(s):

Panagiota Karagianni ◽

Larbi Amazit ◽

Jun Qin ◽

Jiemin Wong

Keyword(s):

Transcriptional Repression ◽

Mammalian Cells ◽

Specific Binding ◽

Binding Protein ◽

Biological Effects ◽

Binding Activity ◽

Effector Proteins ◽

Heterochromatin Formation ◽

Protein Ubiquitination

ABSTRACT Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

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Immunoaffinity purification of juvenile hormone-binding protein from Galleria mellonella hemolymph.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4456 ◽

1996 ◽

Vol 43 (4) ◽

pp. 603-610 ◽

Cited By ~ 12

Author(s):

E Wieczorek ◽

J M Parkitna ◽

J Szkudlarek ◽

A Ozyhar ◽

M Kochman

Keyword(s):

Juvenile Hormone ◽

Galleria Mellonella ◽

Binding Protein ◽

Gel Filtration ◽

Binding Activity ◽

Hormone Binding ◽

Simple Method ◽

Juvenile Hormone Binding Protein ◽

Hormone Binding Protein ◽

Sepharose 4B

Previously described methods of purification of hemolymph juvenile hormone-binding protein (hJHBP) from Lepidoptera were tedious and required multiple steps. These methods resulted in low protein yield (Kramer et al., 1976; Goodman et al., 1978; Peterson et al., 1982; Park et al., 1993; Ozyhar & Kochman, 1987). In this report a simple method of purification of hJHBP from Galleria mellonella (L.) larvae is described. Monoclonal antibodies against hJHBP were obtained and crosslinked to CNBr-activated Sepharose 4B. The hemolymph of G. mellonella was centrifuged and then chromatographed on Sephadex G-200 gel filtration column. Juvenile-hormone-binding activity containing material from Sephadex G-200 column was subjected to purification on an immunoaffinity column. Bound protein was eluted from anti-hJHBP Sepharose 4B gel by lowering pH to 3.0 with 200 mM citric acid 200 mM Na2HPO4 buffer. This method resulted in 320-fold purification of G. mellonella hJHBP with 56% yield.

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