Binding of centchroman—a nonsteroidal antifertility agent to human plasma proteins

Summary Centchroman, a non-steroidal antifertility agent showed a low affinity (K_d=13.19×10^–6 M) and nonsaturable binding to human plasma. Centchroman did not complete either with sex hormone binding globulin or corticosteroid binding globulin. Polyacrylamide gel electrophoresis and temperature dependent binding characteristics revealed that the protein responsible for centchroman binding to human plasma resembles albumin.Acknowledgment. The authors are grateful to Dr. Nitya Nand for his interest in this investigation. CDRI Communication No. 3333.     

Binding capacity of the testosterone-binding plasma protein in 2 wild mammals with cyclic testicular activity, the fox and badger

The binding capacity of the plasma testosterone binding globulin (TeBG) as determined by the equilibrium dialysis method, in fox and badger, was shown to wary in relation to the seasonal endocrine activity of the testis. During the winter breeding period, the TeBG binding capacity was low in the fox and high in the badger, whereas an opposite situation prevailed during the sexual quiescence phase. Independent of those seasonal variations, the binding capacity of TeBG was always lower in fox than in badger.     

Sexual differences in the development of plasma thyroxine levels in the embryo and young chick]

Plasma thyroxine (T4) levels were separately ascertained in male and female embryo and young chicken from the 12th day of incubation till the 4th day after hatching by the thyroxine binding globulin technique. In both sexes, plasma T4 reach a peak the 20th day of incubation, but values are significantly higher in females. A sharp decrease occurred thereafter, plasma T4 tending toward adult values the 4th day after hatching.     

Binding of (125I) triiodothyronine to human peripheral leukocytes and its internalization

Ultrastructural autoradiography showed high specific binding of (125I) triiodothyronine, as confirmed by a competition test, to plasma membranes, nuclei and mitochondria of human peripheral leukocytes. A high level of binding was also noted on the granulocytes' granules, especially in eosinophils.     

On the binding of steroid sulfates to albumin

3H-Labeled steroid sulfates, sulfate of estrone (E1S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E1S). The apparent equilibrium constants (K) of the tracers did not measurably change at concentrations of the non-radioactive sulfates below 10(-5) mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of 3H-E1S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E1S in vivo, because of its preferential occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.     

Binding of (125I) triiodothyronine to human peripheral leukocytes and its internalization

Summary Ultrastructural autoradiography showed high specific binding of (¹²⁵I) triiodothyronine, as confirmed by a competition test, to plasma membranes, nuclei and mitochondria of human peripheral leukocytes. A high level of binding was also noted on the granulocytes' granules, especially in eosinophils.     

Plasma thyroxine levels in Duchenne muscular dystrophy

Some young Duchenne muscular dystrophy (DMD) patients (3-7 years) had total thyroxine (T4) levels and T4 to thyroxine binding globulin (TBG) ratios above the normal range and significantly increased free thyroxine indices (fT4I) which, however, remained within the normal range. Older DMD patients (7-11 years) had T4 and TBG levels and fT4I similar to normal. In both DMD groups the thyroxine binding index (TBI) values were in the normal range.     

In vivo and in vitro differences between leukocytic uptake of oligodeoxynucleotides

Development and application of therapeutic oligonucleotides rely on proper analysis of binding and uptake. We have used several model oligodeoxynucleotides (ODNs) to analyze binding/uptake by rat and human leukocytes. Here we describe: (1) differences between in vivo and in vitro uptake of ODNs to rat leukocytes, (2) differences after injection of lipopolysaccharide (LPS), (3) large in vitro differences between primary mononuclear cells in PBS, plasma and blood, and (4) differences of ODN uptake between rat and human leukocytes. Our data show that ODN uptake by primary blood cells was different in PBS, plasma and blood. In addition, LPS treatment increased ODN uptake by leukocytes in blood, indicating that pathological conditions may influence ODN uptake. Furthermore, ODN uptake in rat and human blood is also different, suggesting that preclinical ODN uptake data from rat blood cannot easily be extrapolated to the human condition. Received 17 December 2007; received after revision 16 January 2008; accepted 5 February 2008     

Interaction of epidermal growth factor with human fibroblasts in culture: an autoradiographic study at the ultrastructural level]

The potent mitogen epidermal growth factor (EGF) binds to specific receptors on human fibroblasts. In the present study we have used a quantitative EM autoradiographic approach to visualize the events involved in the binding process. When 125I-EGF is incubated at 4 degrees C for 120 min, labeled EGF primarily localizes to the plasma membrane of the fibroblast but when incubated at 37 degrees C for 120 min., over 2/3 of the labeled material is internalized by the cell. The internalized radioacitivity is primarily localized to lysomes. These studies demonstrate a temperature-dependent internalization of EGF following initial binding to specific plasma membrane receptors.     

Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.     

Vasoactive intestinal peptide (VIP): specific receptors and adenylate cyclase activation in a human prolactin-secreting pituitary tumor]

Receptors for the Vasoactive Intestinal Peptide (VIP) were characterized in particles enriched in plasma membranes obtained from a human prolactin-secreting pituiatry tumor. Native VIP inhibited competitively the binding of 125I-VIP to the particles and stimulated cyclic AMP production; both these effects were observed at concentrations of VIP as low as 10(-11)-10(-10) M, which are compatible with VIP concentrations in the hypothalamopituitary portal blood.     

An electrophoretic investigation of the binding of 3-14C coumarin to rat serum proteins.

The binding of coumarin to serum proteins of the rat has been demonstrated. Of the total bound coumarin (37% of injected dose), 36% was bound to slow and fast oc1 globulins, 11% to the post albumins, 10% to globulin and 9% to albumin.     

Purification from human plasma of endogenous sodium transport inhibitor(s)

Na+,K+-ATPase inhibitors extracted from plasma of healthy human subjects displaced 3H-ouabain binding to human erythrocytes and inhibited the Na+ efflux catalyzed by the Na+,K+-pump and unexpectedly the Na+,K+-cotransport system without alteration of the Na+,Na+-exchange or the Na+ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.     

How sphingolipids bind and shape proteins: molecular basis of lipid-protein interactions in lipid shells,rafts and related biomembrane domains

Understanding the molecular mechanisms controlling the association of proteins with lipid rafts is a central issue in cell biology and medicine. A structurally conserved motif (the 'sphingolipid binding domain') has been characterized in unrelated cellular and microbial proteins targeted to lipid rafts. I propose that the structuration of a sphingolipid shell around the sphingolipid binding domain not only extracts the protein from the liquid-disordered phase of the plasma membrane, and ensures its delivery to lipid rafts, but also influences its conformation. The chaperone activity of sphingolipids in shells and rafts may play an important role in infectious and conformational diseases(human immunodeficiency virus-1, prions, Alzheimer).     

Plasma thyroxine levels in Duchenne muscular dystrophy

Summary Some young Duchenne muscular dystrophy (DMD) patients (3–7 years) had total thyroxine (T₄) levels and T₄ to thyroxine binding globulin (TBG) ratios above the normal range and significantly increased free thyroxine indices (fT₄I) which, however, remained within the normal range. Older DMD patients (7–11 years) had T₄ and TBG levels and fT₄I similar to normal. In both DMD groups the thyroxine binding index (TBI) values were in the normal range.     

On the binding of steroid sulfates to albumin

Summary ³H-Labeled steroid sulfates, sulfate of estrone (E₁S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E₁S). The apparent equilibrium constants (K) of the tracers did not measuraby change at concentrations of the non-radioactive sulfates below 10^–5 mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of³H-E₁S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E₁S in vivo, because of its preferetial occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.Acknowledgment. This investigation received financial support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organisation, and from the Ford Foundation.     

In vitro amiodarone protein binding and its interaction with warfarin

The binding of amiodarone to human plasma protein and to bovine serum albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Immunochemical evidence for the specific binding of putrescine to plasma proteins including fibronectin

Human plasma contains proteins capable of binding 14C putrescine by the action of Ca++ activated transglutaminase. These proteins have molecular weights from 32 to 220 K and above. One of these (with a molecular weight of 220 K) has been identified as fibronectin by the use of an antifibronectin antiserum. Evidence for a protein with a molecular weight identical to that of fibronectin has been obtained on PAGE analysis of the precipitate formed on incubating human serum with antipolyamine antiserum.     

In vitro amiodarone protein binding and its interaction with warfarin

Summary The binding of amiodarone to human plasma protein and to bovine serum, albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.This work was partially funded by the CNR (National Research Council, Rome, Italy), Program on Clinical Pharmacology and Rare Diseases. The authors would like to thanks Drs E. Marzi and E. riva for their help.