Demonstration of specific dopamine receptors on human pituitary adenomas

1987 ◽  
Vol 114 (4) ◽  
pp. 595-602 ◽  
Author(s):  
Masafumi Koga ◽  
Haruyoshi Nakao ◽  
Masayo Arao ◽  
Bunzo Sato ◽  
Keizo Noma ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Binding Sites ◽  
Pituitary Adenomas ◽  
Binding Capacity ◽  
Affinity Binding ◽  
Human Pituitary ◽  
Human Pituitary Adenoma ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Dissociation Constant Kd

Abstract. Dopamine receptors on human pituitary adenoma membranes were characterized using [3H] spiperone as the radioligand. The specific [3H]spiperone binding sites on prolactin (PRL)-secreting adenoma membranes were recognized as a dopamine receptor, based upon the data showing high affinity binding, saturability, specificity, temperature dependence, and reversibility. All of 14 PRL-secreting adenomas had high affinity dopamine receptors, with a dissociation constant (Kd) of 0.85 ±0.11 nmol/l (mean ± sem) and a maximal binding capacity (Bmax) of 428 ± 48.6 fmol/mg protein. Among 14 growth hormone (GH)-secreting adenomas examined, 8 (57%) had dopamine receptors with a Kd of 1.90 ± 0.47 nmol/l and a Bmax of 131 ± 36.9 fmol/mg protein. Furthermore, 15 of 24 (58%) nonsecreting pituitary adenomas also had dopamine receptors with a Kd of 1.86 ± 0.37 nmol/l and a Bmax of 162 ± 26.0 fmol/mg protein. These results indicate that some GH-secreting adenomas as well as some nonsecreting pituitary adenomas contain dopamine receptors. But their affinity and number of binding sites are significantly lower (P < 0.05) and fewer (P < 0.001) respectively, than those in PRL-secreting adenomas.

Interfaces of Molecular Recognition Sites and Biosensors; Novel Bio-Sensing Materials

2015 ◽  
Vol 1793 ◽  
pp. 1-6
Author(s):  
Kyung M. Choi
Keyword(s):  
Binding Sites ◽  
Glass Surface ◽  
High Sensitivity ◽  
Affinity Binding ◽  
Sensing Element ◽  
Practical Applications ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Number Of Binding Sites ◽  
Low Sensitivity

ABSTRACTWe introduce a sensing element, “Molecularly Imprinted Polymer (MIP),” which created by “Molecular Imprinted Technique.” However, the sensitivity of MIP’s based bio-sensors limits for practical applications due to the low sensitivity. To achieve a high sensitivity of MIP’s based sensors, the synthesis of “high affinity receptor or binding sites,” such as “monoclonal particles” is a key objective. In previous studies, affinity distribution plots indicated that “high affinity binding sites” were obtained when the number of binding sites per particle decreased. It means that smaller particles are expected to have higher affinity binding sites compared to larger particles. The result motivated us to produce small-sized MIP’s particles for the achievement of higher sensitivity. Microfluidic Synthesis has taken a great attention to synthesize small particles. However, the microfluidic synthesis gave us a difficulty, especially collections of MIP’s particles from the surface of PDMS-based microchannels due to a sticking problem. Thus, we employed a new approach, which can collect MIP’s particles without any sticking problem from the surface of the reactor. It is a photopatterned MIP’s system generated on the glass surface. We prepared a photomask with micro-sized patterns and then fabricate MIP’s particles on a glass surface by photopolymerization. Uniform MIP’s patterns were printed on the glass surface. The interface between the glass surface and the MIP’s pattern was observed by SEM. Micro-sized MIP’s particles were collected from the glass surface by scratching off the photocured MIP’s patterns.

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons

1991 ◽  
Vol 261 (6) ◽  
pp. C1048-C1054 ◽  
Author(s):  
L. G. Meszaros ◽  
P. Volpe
Keyword(s):  
Binding Sites ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Ruthenium Red ◽  
Hill Coefficient ◽  
Stopped Flow ◽  
High Affinity ◽  
Protein Dissociation ◽  
Dissociation Constant Kd ◽  
Slight Inhibition

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

The ‘ligand-induced conformational change’ of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region

1998 ◽  
Vol 111 (12) ◽  
pp. 1759-1766 ◽  
Author(s):  
J. Tsuchida ◽  
S. Ueki ◽  
Y. Takada ◽  
Y. Saito ◽  
J. Takagi
Keyword(s):  
Conformational Change ◽  
Binding Sites ◽  
K562 Cells ◽  
Affinity Binding ◽  
Fab Fragment ◽  
High Affinity Binding ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Beta 1 Integrin ◽  
Beta1 Subunits

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.

Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei

1990 ◽  
Vol 258 (5) ◽  
pp. C933-C943 ◽  
Author(s):  
A. A. Altamirano ◽  
B. A. Watts ◽  
J. M. Russell
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Affinity Constant ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Relative Affinity ◽  
Microsomal Membranes ◽  
Loligo Pealei ◽  
Dissociation Constant Kd

Saturable high-affinity binding of [3H] bumetanide [dissociation constant (KD) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (Bmax) was approximately 6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and depended on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) approximately 1 microM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.

Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

1988 ◽  
Vol 255 (6) ◽  
pp. E912-E919 ◽  
Author(s):  
R. S. Haber ◽  
J. N. Loeb
Keyword(s):  
Thyroid Hormone ◽  
Atpase Activity ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Ouabain Binding ◽  
High Affinity ◽  
Mammalian Tissues ◽  
Dissociation Constant Kd ◽  
Crude Membrane Fraction

The administration of thyroid hormone is known to result in an induction of the Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na+-K+-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain, we have studied the effects of 3,3',5-triiodo-L-thyronine (T3) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na+-K+-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (KIs) for ouabain of approximately 10(-7) and 10(-4) M, respectively. Treatment of hypothyroid rats with T3 (50 micrograms/100 g body wt on 3 alternate days) nearly tripled that portion of the Na+-K+-ATPase activity corresponding to the high-ouabain-affinity form (increased by 178 +/- 24%), whereas the enzyme activity corresponding to the low-ouabain-affinity form was only slightly changed (increased by 20 +/- 5%). Measurement of the specific binding of [3H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (Kd) of slightly less than 10(-7) M, whose maximal binding capacity was increased by T3 treatment by 185%. The calculated catalytic turnover associated with the high-affinity site was 70-80 molecules ATP hydrolyzed.site-1.s-1 and was unchanged by T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity calcium binding sites in luminal and basolateral renal membranes

1985 ◽  
Vol 248 (4) ◽  
pp. F472-F481
Author(s):  
Z. Talor ◽  
G. Richison ◽  
J. A. Arruda
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Ruthenium Red ◽  
Affinity Constant ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Ca Uptake ◽  
Number Of Binding Sites

We evaluated Ca binding by highly purified luminal (L) and basolateral (BL) tubular membranes prepared from beef kidney. Ca binding was measured by using 45Ca and a rapid-filtration technique. After Ca uptake reached equilibrium, the vesicles were lysed and the amount of 45Ca retained in the membranes was considered the bound Ca. Ca binding in both membranes accounted for approx. 80% of total Ca uptake. Analysis of binding data by Scatchard plot revealed the presence of two distinct types of binding sites in both L and BL membranes. The high-affinity binding sites showed a similar affinity constant of 10(-5)M for both L and BL membranes, but the maximum number of binding sites was 0.75 and 1.6 nmol/mg protein, respectively. In contrast, the low-affinity binding sites were similar regarding affinity constant and maximum number of binding sites in the two membranes. In L and BL membranes, high-affinity binding sites were selective for Ca, as high concentrations of divalent cations were required to inhibit Ca binding. In both membranes Ca binding was inhibited by ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP. These results demonstrate that L and BL membranes possess high-affinity binding sites with different capacities but similar characteristics as regards affinity constant and stimulation and inhibition of binding. The data further demonstrate that most of Ca uptake by these membranes represents binding.

scholarly journals Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1102-1108
Author(s):  
P Gascard ◽  
CM Cohen
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
High Capacity ◽  
Affinity Binding ◽  
Similar Extent ◽  
High Affinity ◽  
Principal Effect ◽  
Glycophorin C ◽  
Inside Out

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

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