scholarly journals Effects of aging on the synthesis of antithrombin-binding sites on heparin chains and heparan sulphate chains in the rat

1995 ◽  
Vol 312 (1) ◽  
pp. 245-249
Author(s):  
A A Horner
Keyword(s):  
Mast Cells ◽  
Binding Sites ◽  
Heparan Sulphate ◽  
Significant Decline ◽  
Male Rats ◽  
High Affinity ◽  
Peritoneal Mast Cells ◽  
Effects Of Aging ◽  
Age Range

[35S]Heparin proteoglycans were isolated from the skins and peritoneal mast cells of male rats aged 2 to 22 months. Their [35S]heparin chains were separated on antithrombin-agarose into fractions with high and low affinities for antithrombin. In skin, the proportion of 35S-labelled high-affinity heparin chains declined from 23% at 2 months to 8% at 12 months and did not change significantly between 12 and 22 months. In peritoneal mast cells, the proportion of 35S-labelled high-affinity heparin chains increased from 14% at 2 months to 21% at 4 months and then did not vary significantly until 15 months of age. By 21 months a consistent and significant decline to 8% occurred. The structures of high-affinity heparin proteoglycans did not change with age. Their decreased proportions, without change in their structure, may indicate that they are produced by a unique subset of mast cells, the proportion of which declines with age. [35S]heparan sulphate chains were isolated from skins and brains of rats in the same age range and fractionated on antithrombin-agarose. There were no significant variations in the proportions of 35S-labelled high-affinity heparan sulphate chains in skin (10%) or brain (24%) between 4 and 22 months of age.

scholarly journals Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity

1984 ◽  
Vol 222 (3) ◽  
pp. 639-647 ◽  
Author(s):  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Sites ◽  
Maximal Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Male Rats ◽  
Heart Mitochondria ◽  
Binding Characteristics ◽  
Functional Sites ◽  
High Affinity ◽  
Malonyl Coa

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5′-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan

1994 ◽  
Vol 299 (2) ◽  
pp. 507-513 ◽  
Author(s):  
G Pejler ◽  
K Söderström ◽  
A Karlström
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Binding Sites ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Macromolecular Complex ◽  
Mast Cell Protease ◽  
Peritoneal Mast Cells ◽  
Terminal Amino

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.

PHYSIOLOGICAL VARIATION IN ABUNDANCE OF OESTROGEN SPECIFIC HIGH-AFFINITY BINDING SITES IN HYPOTHALAMUS, PITUITARY AND UTERUS OF THE RAT

1975 ◽  
Vol 64 (3) ◽  
pp. 443-449 ◽  
Author(s):  
M. GINSBURG ◽  
N. J. MACLUSKY ◽  
I. D. MORRIS ◽  
P. J. THOMAS
Keyword(s):  
Binding Sites ◽  
Physiological State ◽  
Oestrous Cycle ◽  
Female Rats ◽  
Male Rats ◽  
Affinity Binding ◽  
Equilibrium Dissociation Constant ◽  
High Affinity Binding ◽  
High Affinity ◽  
Equilibrium Dissociation

SUMMARY High-affinity binding of [2,4,6,7-3H]oestradiol-17β has been studied in cytosols prepared from hypothalami, pituitaries and uteri of female rats killed at different stages of the oestrous cycle, and in cytosols prepared from the hypothalami and pituitaries of male rats. In all cases the equilibrium dissociation constant of reaction was of the order of 10−10 mol/l. The number of available high affinity sites per tissue (n) varied with physiological state. In females, n fluctuated with the oestrous cycle. In hypothalamus and pituitary, n fell by about 60 and 40% respectively in pro-oestrus, replenishment occurred during oestrus but could be delayed by phenobarbitone administration during the afternoon of pro-oestrus. In the uterus, n varied biphasically, there being peaks during dioestrus and oestrus, and troughs at pro-oestrus and metoestrus. The numbers of available sites at metoestrus were 12·5 × 109, 10·6 × 1010 and 24·4 × 1010 for hypothalamus, pituitary and uterus respectively. In male rats, values for n were similar to those obtained for females at pro-oestrus (n/hypothalamus = 6·8 × 109, n/pituitary = 4·2 × 1010). Binding was oestrogen specific in all the tissues studied.

scholarly journals Gender-Related Effects of Sex Steroids on Histamine Release and FcεRI Expression in Rat Peritoneal Mast Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Samira Muñoz-Cruz ◽  
Yolanda Mendoza-Rodríguez ◽  
Karen E. Nava-Castro ◽  
Lilián Yepez-Mulia ◽  
Jorge Morales-Montor
Keyword(s):  
Mast Cells ◽  
Histamine Release ◽  
Sex Steroids ◽  
Male Rats ◽  
Peritoneal Mast Cells ◽  
High Affinity Receptor ◽  
Ige Mediated ◽  
Affinity Receptor ◽  
And Gender ◽  
Pathologic Processes

Mast cells (MCs) are versatile effector and regulatory cells in various physiologic, immunologic, and pathologic processes. In addition to the well-characterized IgE/FcεRI-mediated degranulation, a variety of biological substances can induce MCs activation and release of their granule content. Sex steroids, mainly estradiol and progesterone, have been demonstrated to elicit MCs activation. Most published studies have been conducted on MCs lines or freshly isolated peritoneal and bone marrow-derived MC without addressing gender impact on MC response. Our goal was to investigate if the effect of estradiol, progesterone, testosterone, and dihydrotestosterone (DHT) on MCs may differ depending on whether female or male rats are used as MCs donors. Our results demonstrated that effect of sex steroids on MCs histamine release is dose- and gender-dependent and can be direct, synergistic, or inhibitory depending on whether hormones are used alone or to pretreat MCs followed by substance P-stimulation or upon IgE-mediated stimulation. In contrast, sex steroids did not have effect on the MC expression of the IgE high affinity receptor, FcεRI, no matter female or male rats were used. In conclusion, MCs degranulation is modulated by sex hormones in a gender-selective fashion, with MC from females being more susceptible than MC from males to the effects of sex steroids.

scholarly journals Presence of a high-affinity Ca2+-and Mg2+-dependent ATPase in rat peritoneal mast-cell membranes

1985 ◽  
Vol 226 (1) ◽  
pp. 335-338 ◽  
Author(s):  
L M Amende ◽  
M A Donlon
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Atpase Activity ◽  
Cell Membranes ◽  
Plasma Membranes ◽  
Peritoneal Mast Cell ◽  
High Affinity ◽  
Dependent Atpase ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells

Purified perigranular and plasma membranes isolated from rat peritoneal mast cells were examined for Ca2+- and Mg2+-dependent ATPase activity. Isolated perigranular membranes contained only a low-affinity Ca2+- or Mg2+-dependent ATPase (Km greater than 0.5 mM). The plasma membranes contained both a low-affinity Ca2+- or Mg2+-dependent ATPase (Km = 0.4 mM, Vmax. = 20 nmol of Pi/min per mg), as well as a high-affinity Ca2+- and Mg2+-dependent ATPase (Km = 0.2 microM, Vmax. = 6 nmol of Pi/min per mg).

Interaction of transferrin with rat alveolar macrophages

1995 ◽  
Vol 73 (1-2) ◽  
pp. 73-79
Author(s):  
Maria Janicka ◽  
Erwin Regoeczi ◽  
Maria Bolyos ◽  
Wei-Li Hu
Keyword(s):  
Alveolar Macrophages ◽  
Transferrin Receptor ◽  
Binding Sites ◽  
Heparan Sulphate ◽  
Bovine Lactoferrin ◽  
Heparan Sulphate Proteoglycan ◽  
High Affinity ◽  
Sulphate Proteoglycan ◽  
Order Of Magnitude ◽  
Affinity Interaction

Binding of rat transferrin to isolated alveolar macrophages was investigated in the 0.125 nM to 2 μM range. Computer analysis of the data revealed two classes of binding sites, a small number (<1000 exposed/cell) having high affinity (dissociation constant (Kd), 3.4 nM) and a large number (approximately 4 × 106/cell) having low affinity (Kd48 μM). Measurements with a monoclonal antibody to the rat transferrin (rTf) receptor yielded values in the same range as the high-affinity sites derived from studies of ligand binding. Binding to the low-affinity sites at pH 5.8 was nearly one order of magnitude stronger than that at pH 7.3. Bovine lactoferrin (12 μM), cationized bovine serum albumin (14 μM), L-arginine (50 mM), and L-lysine (50 mM) did not compete against rTf binding to the low-affinity sites. Removal of an average of 2.6 × 108sialyl residues from each cell did not affect binding. Heparan sulphate proteoglycan purified from alveolar macrophages bound strongly to immobilized rTf, thus raising the possibility that the low-affinity interaction of transferrin with these cells may be mediated, at least in part, by this glycosaminoglycan.Key words: heparan sulphate proteoglycan, macrophage, transferrin, transferrin receptor.

Thyrotropin action is impaired in the thyroid gland of old rats

1992 ◽  
Vol 126 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Frédéric Reymond ◽  
Nicole Dénéréaz ◽  
Thérèse Lemarchand-Béraud
Keyword(s):  
Thyroid Gland ◽  
Adenylate Cyclase ◽  
Binding Sites ◽  
Hormone Secretion ◽  
Plasma Concentrations ◽  
Male Rats ◽  
High Affinity ◽  
Young Rats ◽  
Old Rats ◽  
Camp Formation

Aging in rats is characterized by low plasma concentrations of thyroid hormones with unchanged levels of TSH, suggesting an altered TSH action in addition to the impaired regulation of TSH secretion. To evaluate TSH action we determined TSH binding to thyroid membranes of young and old male rats (3–4 and 24–26 months of age), as well as the activity of adenylate cyclase in basal and stimulated conditions. Saturation analyses of [125I]-bTSH to thyroid membranes in the presence of increasing quantities of unlabelled bTSH (0.03–100 mU) show two types of binding sites, one of high affinity (Ka 1.5 109 mol l−1) the other of lower affinity (Ka 1.2 108 mol l−1), which are similar in both age groups. The number of TSH binding sites of high affinity is less in old rats than in young rats (7.6±0.9 vs 14.8±1.1 TSH mU/mg protein, N = 11 and 10 respectively, p< <0.001), whereas the number of binding sites of low affinity is not significantly different (76.0±8.2 vs 99.1±9.0 TSH mU/mg protein). The activity of adenylate cyclase determined in basal conditions is similar in both old and young rats (1.11±0.12 vs 1.04±0.9 nmol cAMP/2 h x mg/protein). TSH (10 mU) induced a significant increase in cAMP formation with the thyroid membranes from young rats but not with those from old rats. In contrast, the stimulation of cAMP formation by GTP (2 mmol/l) or forskolin (10 mmol/l), two direct stimulators of adenylate cyclase, is similar in both groups of rats (200% and 250%, respectively). These data suggest that the reduced action of TSH on the thyroid gland of old rats is due to the decreased number of TSH binding sites, which may also be partly responsible for the low thyroid hormone secretion with aging in spite of the unchanged levels of TSH. A postreceptor defect does not seem to be involved, since direct stimulations of adenylate cyclase by GTP or forskolin are just as effective in old rats as in young rats.

Binding of oestradiol, progesterone and prolactin in rat lung

1983 ◽  
Vol 97 (2) ◽  
pp. 301-310 ◽  
Author(s):  
R. R. Ben-Harari ◽  
Tamar Amit ◽  
M. B. H. Youdim
Keyword(s):  
Binding Sites ◽  
Protein Concentration ◽  
Fetal Lung ◽  
Male Rats ◽  
Adult Rats ◽  
High Affinity ◽  
Surfactant Production ◽  
High Affinity Site ◽  
Number Of Binding Sites ◽  
Ovine Prolactin

Binding of [3H]oestradiol and of [3H]progesterone in the cytosol from lungs of adult male rats was suppressible, dependent on incubation time and on protein concentration and was protein in nature. Suppressible binding of oestradiol consisted of a high affinity site (dissociation constant (Kd), 1 × 10−9±0·2 × 10−9 mol/l; maximum number of binding sites (Bmax), 0·7±0·2 pmol/mg protein) and a lower affinity site (Kd, 2·4× 10−8±0·6 × 10−8 mol/l; Bmax, 6·3±0·4 pmol/mg protein) and showed evidence of positive co-operation. Suppressible binding of progesterone consisted of a single site with a Kd of 6× 10−9± 1 × 10−9 mol/l and Bmax of 44·5±8 fmol/mg protein. Binding of 125I-labelled ovine prolactin was found in homogenates of fetal lung (20 days of gestation) but not of adult lung (80 days of age). Treatment of adult rats with ovine prolactin was associated with an increase in the number of binding sites of high affinity for 125I-labelled ovine prolactin but these sites showed an altered specificity. This 'up-regulation' of the prolactin binding may provide a mechanism by which prolactin stimulates surfactant production in lung. These results, together with the known effects of these hormones on certain lung functions, provide further evidence that lung is a target organ for oestradiol, progesterone and prolactin.

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