Specific Cholesterol Binding Drives Drastic Structural Alterations in Apolipoprotein A1.

The α1-adrenoceptor is considered to regulate micturition in patients with benign prostatic hypertrophy. This study demonstrated the specific binding sites of [3H]-bunazosin hydrochloride (4-amino-2-2-[4-butanoyl-hexahydro-1H-1, 4-diazepin-1-yl]-6, 7-dimethoxy quinazoline hydrochloride) in the large sections of the prostate. Eleven surgical specimens of en bloc retropubic prostatectomy were evaluated by in vitro receptor autoradiography. Autoradiograms were assessed using a computerized image analysis system to locate the specific binding sites. Suitable specimens were coated with emulsion for microautoradiography. Specific binding sites were recognized mainly in the perinodular regions of the prostate as well as in some nodules and in the urethral region. Silver grains were recognized in the fibromuscular stroma of the perinodular and subepithelial regions of the prostatic ducts and acini. Some epithelial cells and the basement membrane of the ducts, the acini, and the prostatic urethra also revealed the accumulation of silver grains. Whether this latter finding indicated specific binding to α1-adrenoceptors or not is a matter for further investigation. This study indicated that [3H]-bunazosin was useful for the qualitative demonstration of specific α1-adrenoceptor binding sites in the prostate by in vitro receptor autoradiography.

Beta-adrenergic receptor in the brain: Comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates

M-ORBIS is a Molecular Cartography approach that performs integrative high-throughput analysis of structural data to localize all types of binding sites and associated partners by homology and to characterize their properties and behaviors in a systemic way. The robustness of our binding site inferences was compared to four curated datasets corresponding to protein heterodimers and homodimers and protein–DNA/RNA assemblies. The Molecular Cartographies of structurally well-detailed proteins shows that 44% of their surfaces interact with non-solvent partners. Residue contact frequencies with water suggest that ∼86% of their surfaces are transiently solvated, whereas only 15% are specifically solvated. Our analysis also reveals the existence of two major binding site families: specific binding sites which can only bind one type of molecule (protein, DNA, RNA, etc.) and polyvalent binding sites that can bind several distinct types of molecule. Specific homodimer binding sites are for instance nearly twice as hydrophobic than previously described and more closely resemble the protein core, while polyvalent binding sites able to form homo and heterodimers more closely resemble the surfaces involved in crystal packing. Similarly, the regions able to bind DNA and to alternatively form homodimers, are more hydrophobic and less polar than previously described DNA binding sites.

The adenine nucleotide translocation in mitochondria has previously been established as an exchange between exogenous and endogenous adenine nucleotides across the inner membrane. The specificity and the control of the exchange are examined with the following major results: The adenine nucleotide translocation is relatively specific for exogenous ADP and ATP, AMP being nearly inactive. Among other nucleotides tested, only dADP and dATP exchange with a noticeable activity. In the controlled state ADP exchanges 2–4 times faster than ATP. If simultaneously added, ADP and ATP compete for the exchange, with ADP being about tenfold more active than ATP. The specificity of the exit of adenine nucleotides in the exchange is similar to the specificity of the entrance with the difference that ADP and ATP are released with equal activity in proportion to their intramitochondrial content. AMP is released only after a slow conversion to ADP. Therefore the short time exchange is limited by the endogenous content of ADP plus ATP. The exchange is influenced by the metabolic state of the mitochondria. The ATP exchange is more variable than the ADP exchange. Two effects are elucidated: (a) the influence of the metabolic state on the relative content of AMP which inhibits both the ADP and ATP exchange (b) the coupling of the energy transfer system which inhibits only the ATP exchange. An example for case (a) is the inhibition of the ADP and ATP exchange by arsenate and an example for case (b) is the strong increase of the ATP exchange on uncoupling. The following effects are relevant to the mechanism of the control of the exchange by ATP. The stimulation of the ATP exchange by uncoupler has the same concentration dependence as the uncoupling of oxidative phosphorylation (Km [CCP] = 0.08 μM, where CCP = carbonyl‐cyanide‐phenylhydrazone). Oligomycin does not abolish the uncoupler effect on the ATP exchange. “Endogenous uncoupling” on aging of mitochondria also stimulates the ATP exchange. Valinomycin plus K+ only slightly stimulate the ATP exchange. Anaerobiosis stimulates the ATP exchange to a smaller extent than uncoupling.In competition with ADP the effects of energy transfer on ATP exchange are more strongly revealed. On uncoupling the more than tenfold preference for ADP is fully abolished. It is concluded that basically the exchange for ADP and ATP has equal specificity in forward and reverse reaction. In the controlled state a superimposed force makes the specificity asymmetric and inhibits the entrance of ATP. This control of the ATP exchange is concluded to be based on the anionic character of the adenine nucleotides. Thus the ATP4‐ex–ADP3‐in exchange is inhibited unless the charge difference is compensated for by an uncoupler stimulated H+ movement across the membrane. Furthermore an electric potential gradient appears to be effective in the controlled state which is abolished on uncoupling.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.

Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study

Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria

Cellular distribution of prostacyclin synthase and specific prostacyclin binding sites in bovine corpora lutea of pregnancy

Interfacial binding sites for cholesterol on GABAA receptors and competition with neurosteroids

Exogenously administered alpha-bungarotoxin binds to embryonic chick spinal cord: Implications for the toxin-induced arrest of naturally occurring motoneuron death

The localization of voltage-sensitive sodium channels was investigated in mouse, rat and rabbit sciatic nerves using iodinated alpha- and beta-Scorpion toxins (ScTx) as specific probes. Saturable specific binding for a beta-ScTx was detected in mouse sciatic nerve homogenates (Kd = 90 pM, binding site capacity = 90 fmol mg-1 protein). LM autoradiographic studies demonstrated that the two types of ScTx stained the Ranvier nodes of the myelinated fibres, and also showed a clear but weaker labelling of the unmyelinated Remak bundles. In the sciatic nerve, which is widely considered as a model 'myelinated nerve', the nodal membrane represented only a small fraction of the total axonal membranes (0.2% and 0.05% for mouse and rabbit sciatic nerves respectively). Therefore, despite their high channel density, nodal membranes contribute only a small proportion of the total labelling by beta-ScTx (15% and 2.3% for mouse and rabbit sciatic nerves respectively), with the major contribution to labelling arising from unmyelinated axons. The distribution of specific binding sites for a beta-Scorpion toxin was then analysed in cross-sections of rabbit sciatic nerve at the EM level. The quantitative analysis of autoradiograms involved three methods, the 50% probability circle method, and two cross-fire analyses using either systematically distributed hypothetical sources or hypothetical sources only located on the plasma membranes of axons and of Schwann cells associated with unmyelinated Remak bundles. No specific beta-Scorpion toxin binding sites were detected at the plasma membrane of Schwann cells from either myelinated fibres or unmyelinated bundles, or at the internodal surface of myelinated axons. Sites were only detected at the surface of unmyelinated axons and at nodal axolemma. Their density in unmyelinated axons was found to be in the range of 1-6 per micron2 of plasma membrane surface area by combining quantitative EM autoradiography and stereological measurements.

Crosslinking/MS Studies of Cholesterol Interactions with Human α1 Glycine Receptor

The effect of the disulfide reducing agent dithiothreitol (DTT) and other thiols on binding of the beta-adrenoceptor antagonist (-)-125iodocyanopindolol (125ICYP) to human mononuclear leucocytes (MNL) was investigated. Saturation experiments and dissociation kinetics revealed two classes of specific 125ICYP binding sites, one of high and the other of low affinity, respectively. In intact MNL DTT caused a decrease in specific binding. This was due almost selectively to a decrease in the affinity of high affinity binding sites, which decreased gradually in a concentration-dependent manner to the affinity of low affinity binding sites. In MNL membranes DTT decreased not only the affinity but also the number of high affinity binding sites. The DTT effect was completely reversible by simple reoxidation on air. The structural isomers (+/-)-DTT. (-)-DTT and dithioerythritol revealed identical effects on specific binding, whereas the monothiols mercaptoethanol and alpha-monothioglycerol, having a lower redox potential, were considerably less effective. In the same concentration range that influenced specific binding. DTT stimulated intracellular cAMP production. These results suggest functionally important disulfide bridges which regulate the affinity of beta-adrenoceptor binding sites in human MNL. They stabilize the receptor in a high affinity state; their reduction causes the conversion of the high affinity state into a low affinity state in a process associated with stimulation of adenylate cyclase. Available evidence indicates that a similar transformation is made by beta-adrenoceptor agonists. Consequently low affinity 125ICYP binding sites preexistent in untreated cells could represent a reduced receptor state resulting from agonist-receptor interaction in vivo.

Two peptides with natriuretic and diuretic properties consisting of amino acids (a.a.) 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF), which normally circulates in humans and animals, were investigated to determine if they have specific binding sites on distal nephrons, proximal tubules, renal cortical and medullary membranes. Competitive binding experiments revealed that proANFs 1-30, 31-67, and 99-126 (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants (Kd) for proANFs 1-30, 31-67, and 99-126 binding to renal cortical membranes were similar at 9.8, 5.1, and 4.7 nM, respectively. In renal medullary membranes, on the other hand, proANF1-30 (Kd = 3.7 nM) and proANF31-67 (Kd = 2.9 nM) had a 10-fold higher binding affinity than ANF (Kd = 56 nM). All three peptides had very weak binding to proximal tubules with the respective Kds for proANF1-30, proANF31-67, and ANF of 892, 789, and 550 nM indicating a 50- to 100-fold less affinity for their binding sites in proximal tubules than in distal nephrons (Kds of 4.9, 5.3, and 11 nM, respectively). Each peptide bound to the respective kidney membranes or tubules between 10(-8) and 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7) M. Neither insulin, growth hormone, nor adrenocorticotrophic hormone competitively inhibited the binding of proANF 1-30, proANF 31-67 or ANF. These results suggest that proANF 1-30 and proANF31-67 do not work through the ANF receptor but rather have their own separate and distinct receptors to mediate their diuretic and natriuretic effects in the kidney.

The insulin-like growth factors (IGFs) are considered important regulators of bone metabolism affecting a number of biologic responses in vitro. Primary fetal rat calvarial cells (PRC) and a cloned adult rat calvarial cell line (C3) both exhibit a concentration-dependent IGF stimulation of [3H]thymidine incorporation into DNA, but the C3 cells show a greater sensitivity and magnitude of response. IGF-I and IGF-II were nearly equipotent in PRC cultures, but IGF-I was more than twice as active as IGF-II in the C3 cultures. This effect of the IGFs on DNA synthesis in two bone cell cultures with different culture histories has been correlated with receptor and binding protein profiles. Specific high-affinity IGF binding sites were found in both cell types. In general, the sites present on PRC cells showed a preference for binding IGF-II over IGF-I, but C3 cells displayed two types of relatively specific binding sites. In both cell types [125I]IGF-I bound primarily to a protein with IGF type I receptor characteristics. However, in PRC cells, [125I]IGF-II cross-linked specifically with proteins that had IGF type II receptor characteristics plus several sites unique to these cells; in C3 cells, [125I]IGF-II bound to a 139 kD protein that could be displaced by either IGF-I or IGF-II. Finally, IGF-II-specific 85 and 67 kD proteins were common to both cell types. From these studies, it is apparent that the IGFs bind to a variety of high-affinity binding sites in bone cells and that these sites differ between a highly responsive and a less responsive bone cell population.