scholarly journals Properties of soluble somatostatin-binding protein

1977 ◽  
Vol 165 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Norio Ogawa ◽  
Tom Thompson ◽  
Henry G. Friesen ◽  
Joseph B. Martin ◽  
Paul Brazeau

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.

1978 ◽  
Vol 56 (1) ◽  
pp. 48-53 ◽  
Author(s):  
N. Ogawa ◽  
T. Thompson ◽  
H. G. Friesen

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.


1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty

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.


1975 ◽  
Vol 53 (6) ◽  
pp. 1135-1140 ◽  
Author(s):  
B. M. Arnold ◽  
M. Kuttner ◽  
D. M. Willis ◽  
A. J. W. Hitchman ◽  
J. E. Harrison ◽  
...  

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.


2009 ◽  
Vol 88 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Gabriela Arevalo-Pinzon ◽  
Hernando Curtidor ◽  
Claudia Reyes ◽  
Martha Pinto ◽  
Carolina Vizcaíno ◽  
...  

1999 ◽  
Vol 13 (3) ◽  
pp. 495-504
Author(s):  
N. E. Erondu ◽  
J. Nwankwo ◽  
Y. Zhong ◽  
M. Boes ◽  
B. Dake ◽  
...  

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.


1990 ◽  
Vol 127 (1) ◽  
pp. 139-148 ◽  
Author(s):  
C. Y. Lee ◽  
D. M. Henricks

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


1977 ◽  
Vol 85 (2) ◽  
pp. 256-266 ◽  
Author(s):  
Valerie Anne Galton

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.


1990 ◽  
Vol 270 (3) ◽  
pp. 577-582 ◽  
Author(s):  
R C Angel ◽  
J A Botta ◽  
R D Morero ◽  
R N Farias

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.


2007 ◽  
Vol 28 (2) ◽  
pp. 705-717 ◽  
Author(s):  
Panagiota Karagianni ◽  
Larbi Amazit ◽  
Jun Qin ◽  
Jiemin Wong

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.


1996 ◽  
Vol 43 (4) ◽  
pp. 603-610 ◽  
Author(s):  
E Wieczorek ◽  
J M Parkitna ◽  
J Szkudlarek ◽  
A Ozyhar ◽  
M Kochman

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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