A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species

The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Specific high-affinity binding sites for a synthetic gliadin heptapeptide on human peripheral blood lymphocytes

The synthetic peptide containing residues 43-49 of alpha-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. We now demonstrate using radiolabeled alpha-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (KD) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of alpha-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of [125I]alpha-gliadin(43-49) than T-lymphocytes. The lymphocyte alpha-gliadin(43-49) receptor may play an important role in mediating the immunological response to alpha-gliadin.     

Specific binding of human C-reactive protein to human monocytes in vitro

The precise biologic function of C-reactive protein (CRP), a major acute phase protein in man, is unknown. The abilities of CRP to bind biologic substrates and to activate the C pathway, and its localization at sites of inflammation argue for an opsonic role for this protein. Such a role has been supported by recent reports of specific binding of CRP to neutrophils. Using highly purified radioiodinated human CRP, we have observed specific binding of this protein to human monocytes in vitro. The binding was reversible and rapid, with a t1/2 for the dissociation reaction of approximately 3 min. Binding was saturable at a CRP concentration of approximately 0.2 microM, with an estimated K from Scatchard analysis of 1.1 x 10(-7) M. Specific binding was calcium-dependent, with optimal binding occurring at calcium concentrations of more than 1.0 mM. No specific binding could be demonstrated to a non-adherent population of mononuclear cells (more than 80% lymphocytes). In other experiments, a 100-fold excess of human IgG failed to inhibit binding, although rabbit CRP produced competitive inhibition of binding which was quantitatively similar to human CRP. The binding was maximal at pH 7.4 and was sensitive to prior trypsin treatment of cells. These studies provide direct evidence for specific binding of soluble human CRP to human monocytes in vitro and thus provide further support for an important functional interaction of this acute phase protein with phagocytic cells in man.     

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

Cerebrospinal fluid and plasma corticotropin-releasing hormone in senile dementia.

Cerebrospinal fluid (CSF) levels of corticotropin-releasing hormone (CRH) and ACTH, and plasma levels of CRH, ACTH and cortisol were determined in samples taken simultaneously from 28 patients with dementia including senile dementia of the Alzheimer type (SDAT), multi-infarct dementia (MID), dementia following a cerebrovascular accident (CVD), and the borderline-to-normal state. CRH levels in CSF were significantly reduced in patients with SDAT and CVD, but not in those with MID, as compared with the borderline cases. ACTH levels in CSF were significantly reduced in the patients with SDAT compared to those with MID. Reduced CRH levels in CSF were found in the patients who showed severe dementia and poor activities of daily living (ADL). Plasma levels of CRH, ACTH and cortisol were normal and were not significantly different among the four groups of patients. CRH levels in CSF were positively correlated with ACTH levels in CSF, but not with the levels of plasma CRH, ACTH or cortisol. Plasma CRH levels were positively correlated with plasma ACTH levels. These results suggest that: 1) abnormalities in the extrahypothalamic CRH system play a role in the pathophysiology of senile dementia, which may not be specific to SDAT; 2) CSF CRH is correlated with the severity of dementia and ADL; 3) the levels of CRH in CSF and plasma are independent, and 4) the plasma CRH reflects, at least in part, the activity of the hypothalamic CRH regulating the secretion of pituitary ACTH.     

Stereoselective binding of disopyramide to human plasma protein

The binding of racemic disopyramide and its two enantiomers to protein were compared in two samples of human plasma, two samples of freshly drawn serum and in a solution of alpha 1-acid glycoprotein. The binding of S(+)-disopyramide was higher at all concentrations as compared to to R(?)-disopyramide, and the binding of the racemate was intermediate. Differences in binding were due to differences in the association constant.     

Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects

Plasma corticotropin-releasing factor (CRF), corticotropin (ACTH) and cortisol levels were simultaneously determined by radioimmunoassays at 0600 h, 1200 h, 1800 h and 2200 h in six normal subjects, in order to examine whether the diurnal rhythm in plasma CRF exists and how it correlates to the diurnal rhythm in plasma ACTH and cortisol concentration. The highest CRF level was observed at 0600 h (7.0 +/- 1.2 pg/ml) and significantly lower levels (p less than 0.01) at 1800 h (1.7 +/- 0.2 pg/ml) and 2200 h (1.9 +/- 0.4 pg/ml). A clear diurnal rhythm was demonstrated in plasma ACTH and cortisol levels, with the highest values at 0600 h (44.6 +/- 8.1 pg/ml and 15.9 +/- 2.0 micrograms/dl, respectively) and the lowest at 2200 h (12.3 +/- 2.8 pg/ml and 4.6 +/- 1.0 micrograms/ml, respectively). These results suggest that the diurnal rhythm in ACTH and cortisol is under the regulation, at least in part, of the diurnal rhythm in CRF secretion.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

Immunoreactive corticotropin-releasing hormone, adrenocorticotropin and cortisol in human plasma during pregnancy and delivery and postpartum

The activity of the hypothalamo-pituitary adrenal axis was examined, by measuring the levels of immunoreactive (IR) corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH) and cortisol (F) in human plasma during normal pregnancy and after delivery with or without complications and during normal postpartum using a specific RIA. The level of IR-CRH in maternal plasma increased progressively during pregnancy, increased further at delivery and declined rapidly to the non-pregnant level on the 1st day postpartum. The level of IR-F in maternal plasma also increased progressively during pregnancy, increased further at delivery, but decreased slowly postpartum, not returning to the non-pregnant level within 5 days. Significant correlations were found between the level of IR-CRH and IR-ACTH, IR-CRH and IR-F, and IR-ACTH and IRF in maternal plasma both during pregnancy and after delivery. It is noteworthy that the concentration of IR-CRH in the maternal plasma at delivery was higher in multiple pregnancy than in normal pregnancy, and that the level of IR-CRH in the umbilical cord in uncomplicated cases was much lower than that in the maternal plasma, and was significantly lower than those in the umbilical cord plasma in cases of asphyxia, IUGR or premature delivery. The level of IR-F, not IR-CRH and IR-ACTH, at normal vaginal delivery was significantly higher than that at elective cesarean section. On these results, we investigated the feto-maternal-hypothalamo-pituitary adrenal axis during pregnancy and delivery, in which CRH plays an important role.     

Absence of plasma protein binding of aldosterone in normal and estrogen-treated rabbit

In experiments on direct effects of prolonged administration of estrogen on In experiments on direct effects of prolonged administration of estrogen on mean arterial pressure (MAP) and plasma corticosteroid-binding variables in the rabbit the following observations were made. Estrogen had no effect on MAP but resulted in a nonsignificant stimulation of total plasma corticosteroids and a marked increase in corticosteroid-binding globulin (CBG) binding capacity which increased from a control value of 18.8 +/- (SD) 1.2 micrograms/100 ml to 28.1 +/- 2.3 micrograms/100 ml (p less than 0.001) following the administration of estrogen for the first 21 days (approx. 10 micrograms/day) and then further to 31.4 +/- 2.8 (p less than 0.001 vs. control values) after a higher estrogen dose of approximately 30 micrograms/day for the next 30 days, respectively. Plasma aldosterone concentration was not affected by estrogen treatment. In contrast to CBG, binding of aldosterone to plasma aldosterone-binding globulin was totally absent before and following the estrogen treatment. The striking difference between the rabbit showing an absence of plasma protein binding of aldosterone and several other animal species is perhaps of great importance for the blood pressure regulation and for understanding of the particular resistance of blood pressure to salt or mineralocorticoids reported in this species.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Levonorgestrel as a new radioligand for determination of sex hormone binding globulin in human plasma

Levonorgestrel which binds with high affinity to SHBG, is suggested as a new radioligand for estimation of SHBG in human plasma. Using 3H-levonorgestrel as a ligand, a number of samples from men, women pregnant and non-pregnant were analysed. The SHBG content was lowest in men, low in women and rose to higher level during pregnancy. The results of the present study suggest that levonorgestrel appears to be a better ligand than dihydrotestosterone for measuring SHBG.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.