Specific Binding Capacity Research Articles

Use of the radioligand [ 125I]-SB-217644 in the characterisation and solubilisation of the novel binding site for the anticonvulsant SB-204269 in rat brain membranes and cell lines

SB-204269 ( trans-(+)-6-acetyl-4 S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2 H-benzo[ b]pyran-3 R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [ 3H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [ 125I]-labelled form of SB-217644 ( trans-6-acetyl-4 S-(3-iodobenzoylamino)-3,4-dihydro-2,2-dimethyl-2 H-benzo[ b]pyran-3 R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [ 125I]-SB-217644 shows a similar binding profile to that of [ 3H]-SB-204269, with a maximum specific binding capacity ( B max) of 286±12 fmol/mg protein, but has twenty-fold higher affinity ( K d value 1.7±0.1 nM). The high affinity and high specific activity of [ 125I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [ 125I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K d value of 2.7±0.3 nM and a B max value of 55±11 fmol/mg protein, with a 7.3±0.3% yield of solubilised binding sites. [ 125I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [ 125I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [ 125I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.

Apolipoprotein C-III can specifically bind to hepatic plasma membranes.

Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of triglyceride-rich lipoproteins by interfering with apo E-mediated receptor clearance of remnant particles from plasma. The mechanism of the interference has not yet been defined. To further explore the role of apo C-III, we first injected mice with 125I-apo C-III. The measurement of radioactivity showed that liver took up 3.3-10 fold as much radioactivity as other organs such as heart, spleen, lung, kidney, stomach, large intestine, small intestine, and muscle. This was confirmed by incubating the tissue homogenates of the organs with 125I-apo C-III that the radiolabeled apo C-III specifically bound to only hepatic homogenate. To investigate which subcellular part or parts of hepatic cells play the role of binding to apo C-III, hepatic cell components of nucleus, mitochondria, microsomes and plasma membranes were then incubated with 125I-apo C-III. The radiolabeled apo C-III could specifically bind to only hepatic plasma membranes. Finally hepatic plasma membranes were purified to study the characteristics of the specific binding with apo C-III. Addition of increasing concentration of 125I-apo C-III to human hepatic plasma membranes revealed saturable binding to membranes with a Kd of 0.31 +/- 0.07 micromol/l. The maximum specific binding capacity was 1.74 +/- 0.45 microg apo C-III/mg membrane protein. In competition studies using unlabeled apo C-III and isolated lipoproteins HDL, LDL and VLDL, only apo C-III and VLDL effectively competed with 125I-apo C-III for membrane binding. The binding of 125I-apo C-III to human liver plasma membranes was Ca2+-independent, and was abolished when plasma membranes were treated with trypsin. The characteristics of 125I-apo C-III binding to mouse liver plasma membranes were similar to those of human liver plasma membranes with the exception of a binding maximum of 1.52 +/- 0.39 microg apo C-III/mg membrane protein. We conclude that apo C-III exhibits high-affinity binding to hepatic plasma membranes, which is saturable, reverse and specific.

Signal transduction pathways in normal human monocytes stimulated by cytokines and mediators: Comparative study with normal human neutrophils or transformed cells and the putative roles in functionality and cell biology

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) -3 induced tyrosine phosphorylation of 92-kDa protein in normal human monocytes. We identified this 92-kDa protein as STAT5, but not as STATs1, 3, and 6 nor c-fes and vav protooncogene products, and demonstrated its translocation to the nucleus, enhancement of specific DNA binding capacity, and potentiation of trancriptional activity by GM-CSF. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) induced tyrosine phosphorylation of 42- and 44-kDa proteins, which were identified as extracellular signal-regulated kinase (ERK), in human monocytes. In marked contrast to neutrophils and MO7e cells, GM-CSF did not induce tyrosine phosphorylation and activation of ERK in monocytes. Among upstream signaling molecules of ERK, Shc was constitutively associated with Grb2 and was not tyrosine-phosphorylated by GM-CSF and FMLP, and Sos1 and c-Raf-1 were not phosphorylated by GM-CSF, IL-3, TNF, and FMLP in monocytes, whereas all these signaling molecules were affected and/or utilized by GM-CSF in MO7e cells. In contrast to neutrophils, p38 was constitutively phosphorylated and agonist-dependent phosphorylation and activation was not detected in human monocytes. Superoxide release stimulated by FMLP was inhibited partially by PD98059 or SB203580, a specific inhibitor of ERK or p38 pathway, and was almost completely inhibited by the combination of both inhibitors, whereas PMA-induced superoxide release was resistant to these two inhibitors in monocytes. PD98059 inhibited GM-CSF-dependent proliferation of MO7e cells. Present results indicate trancriptional roles of STAT5 and functional roles of ERK and/or p38 in normal human monocytes stimulated by physiological receptor-mediated agonists GM-CSF and FMLP. Possible roles of ERK in proliferation of transformed cells were also suggested.

A combined specific target site binding and pharmacokinetic model to explore the non-linear disposition of draflazine.

The capacity-limited high-affinity target site binding of draflazine to the nucleoside transporters located on the erythrocytes is a source of nonlinearity in the pharmacokinetics of the drug. An attractive feature of draflazine is that the specific target site binding characteristics can be determined easily by simultaneously measuring plasma and whole blood concentrations of the drug. Measured drug concentrations following various infusion rates and infusion durations were used to develop a model in which the interrelated blood-plasma distribution, elimination, and specific target site binding of draflazine were incorporated simultaneously. The estimated binding (dissociation) constant Kd was 0.57 ng/ml plasma and the maximal specific erythrocyte binding capacity (BmaxRBC) was 163 ng/ml RBC. The maximal specific binding capacity to the tissues (Bmaxtissue) was estimated to be about 1 mg. The estimated volume of the central compartment (Vplasma + tissue fluids) was 12.9 L and the total intrinsic CL was 645 ml/min. After validation, the model was used to further investigate the impact of the specific high-affinity target site binding of draflazine on its disposition in plasma. The time required to reach steady-state plasma concentrations of draflazine decreased with an increasing infusion rate. Time profiles of the plasma concentrations were not always representative for the time profiles of the specific target site (RBC) occupancy of draflazine, but the t1/2,z in plasma paralleled that of the drug at target sites. The apparent Vd and the t1/2,z decreased with increasing single doses whereas the total CL remained constant. The recovery of draflazine was also dose dependent and increased with increasing doses. Finally, the total CL and apparent Vd of the first dose were greater than those of the second dose of draflazine.

Reducing metabolic rate by immunoneutralisation of catecholamines

A conjugate of noradrenaline and ovalbumin was used to produce anti-noradrenaline antibodies in goats and Brahman steers. Three weeks after the primary immunisation, anti-noradrenaline binding was detected in the sera of 2 of 5 immunised steers but none was detected in any steers 6 weeks after immunisation. Three weeks after immunisation, immunised steers, including the ones with no detectable anti-noradrenaline antibodies, had a larger change (P < 0.02) in fasting metabolic rate from pretreatment values (–14.5 W) than did paired controls (+3.3 W). After 6 weeks, the changes in fasting metabolic rate for the treated and control groups were –17.8 Wand –36.6 W (P < 0.08), respectively. Anti-noradrenaline polyclonal antibodies purified from goat serum had an approximate dissociation constant for binding to noradrenaline of 4 µM and a specific binding capacity of 178 nM in the immunoglobulin solution prepared, equivalent to about 30 mg/L of anti-catecholamine antibodies in the original goat serum. When 1 mL of this immunoglobulin solution was injected into rats, their metabolic rate was lowered by >10% relative to rats treated with control immunoglobulin solution (P < 0.05). These results demonstrate that the lowering of metabolic rate by an immunological approach which targets control points in the sympathetic nervous system is feasible and could offer an immunologically based method of growth promotion for beef cattle.

P21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage.

Cells expressing the R273H mutant of p53, which lacks sequence specific DNA binding capacity, do not undergo cell cycle arrest in G1 following exposure to ionizing or UV radiation because of their inability to induce p21Waf1/Cip1, a cyclin-dependent kinase inhibitor and downstream mediator of p53-dependent DNA damage-induced growth arrest. Following UV-irradiation or treatment with an inhibitor of RNA pol II, we observed a rapid induction of the apoptotic process, as evidenced by DNA fragmentation and the proteolytic cleavage of poly(ADP-ribose) polymerase. Using mimosine, a p21Waf1/Cip1 inducer that bypasses the requirement for transcriptional transactivation by p53, we demonstrated that a G1 cell cycle arrest can prevent apoptosis following UV-irradiation or treatment with an RNA polymerase 11 inhibitor. Serum starvation, which also synchronized cells in G1 but did not induce p21Waf1/Cip1, did not protect cells from apoptosis. These results demonstrate that restoring a late G1 checkpoint by inducing p21Waf1/Cip1 expression can protect cells from DNA damage induced apoptosis. Our results suggest that p21Waf1/Cip1 can interrupt the apoptotic process at a point downstream from p53 accumulation but upstream from caspase-3 activation.

Baculovirus surface display: construction and screening of a eukaryotic epitope library.

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.

Elucidation of a new biological function of an old protein: unique structure of the cobra serum albumin controls its specific toxin binding activity

Although few proteins have been studied as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C 11 and C 502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C 11→F and C 502→T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7±0.2, whereas the latter replacement reduced it to (25±2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C 11 and C 502 in the native CSA that joins the chain ends to form a dense conformation.

Contribution of alpha 1-acid glycoprotein to species difference in lincosamides-plasma protein binding kinetics.

Protein binding kinetics of lincomycin (LM) and clindamycin (CM) were studied using plasma, albumin and alpha 1-acid glycoprotein (AGP) derived from humans, dogs, cattle and sheep. Based on Rosenthal plots of LM and CM, drug-binding property in plasma presented specific and non-specific binding, except for LM in cattle and sheep and for CM in sheep, where only non-specific binding was demonstrated. Dissociation constant (Kd) and binding capacity (Bmax) for specific binding and proportionality constant (PC) for non-specific binding were as follows: Kd = 3.14 mumol/L, Bmax = 15.28 mumol/L, PC = 0.19 for humans; Kd = 3.84 mumol/L, Bmax = 6.55 mumol/L, PC = 0.14 for dogs; PC = 0.12 for cattle; PC = 0.16 for sheep in LM and Kd = 0.94 mumol/L, Bmax = 12.24 mumol/L, PC = 4.98 for humans; Kd = 1.48 mumol/L, Bmax = 9.52 mumol/L, PC = 2.91 for dogs; Kd = 1.22 mumol/L, Bmax = 4.45 mumol/L, PC = 2.40 for cattle; PC = 1.48 for sheep in CM. The specific binding for each species was different, showing more difference in Bmax compared with Kd. The non-specific binding of LM was similar among species whereas that of CM was different, implying species difference. The drug-binding property of AGP for each species was all specific binding and the Kd was comparable to that obtained from plasma, indicating that AGP is a major specific binder in plasma. The lack of detection of specific binding for LM in cattle and sheep and for CM in sheep plasma could be attributable to a higher Kd and lower plasma AGP concentration compared with other species. The drug-binding property of albumin was characterized as all non-specific, without a great difference among species. Except for CM in sheep, the lower PC in albumin solution compared with that in plasma suggested the presence of another non-specific binder in plasma, i.e. lipoprotein. From the simulation of drug-binding percentage to AGP concentrations, AGP could be a major contributor to drug-plasma protein binding in pathological states. The degree of AGP-drug binding for each species could vary according to the degree of increase of AGP concentrations from a healthy to a pathological state, inducing a decrease in the unbound fraction (fp): 6.1 fold for dogs, 4.6 fold for humans, 1.8 fold for sheep and 1.4 fold for cattle in LM; 5.8 fold for dogs, 5.7 fold for cattle, 4.0 fold for humans and 1.5 fold for sheep in CM. Therefore, the disposition and efficacy of lincosamides affected by fp can be modified differently by the change of fp attributable to the alteration of plasma AGP concentration in each species.

Apical expression of functional asialoglycoprotein receptor in the human intestinal cell line HT-29

BACKGROUND & AIMS: The asialoglycoprotein receptor localizes to the basolateral membrane of hepatocytes and to the apical membrane of enterocytes. The aim of this study was to examine HT-29 cells as a polarized cell model for studying apically localized endogenous asialoglycoprotein receptor.METHODS: Subunits H1 and H2 (human) were detected by Western blot and immunoprecipitated using subunit-specific antisera against hepatic receptor peptides. Receptor function was assessed by uptake of iodinated asialo-orosomucoid, immunoglobulin (Ig) A1, and haptocorrin. Immunocytochemistry was analyzed by standard light and confocal microscopy.RESULTS: Receptor content of the minor subunit, H2, was predominant. HT-29 cells mediated specific uptake and degradation of 125I-asialo-orosomucoid. A high-affinity (0.6 x 10(-9) mmol/L) and a low-affinity binding site were present. The specific ligand binding capacity of the apical surface was approximately twice that of the basolateral surface. Immunocytochemistry revealed a predominant apical membrane location of the minor receptor subunit, with some intracellular receptor. The apical H2 subunit was preferentially labeled with amino acid precursors compared with basolaterally located subunit. Human IgA1 bound specifically to HT-29 cells with a molar ratio of 0.26 compared with asialo-orosomucoid; porcine haptocorrin bound with a molar ratio of 1.35.CONCLUSIONS: HT- 29 cells produce a functional apically located asialoglycoprotein receptor and provide a model for receptor trafficking in the enterocyte.(Gastroenterology 1997 Nov;113(5):1501-9)

VRML in Cancer Research: Local Changes in Binding Properties of Wild Type and Mutated p53 Tumor Suppressor Protein

The inactivation of the p53 tumor suppressor function by single missense point mutations is found in almost half of human tumors. Most p53 mutation hotspots are at the DNA binding interface, shown in the three-dimensional (3D) structure of a p53-DNA complex crystallized by Pavletich and coworkers [1]. We have investigated the influence of mutations on the predicted specific DNA binding capacities of p53 by using molecular modeling to compare biochemical properties of wild type and mutated p53 complexed to DNA. Changes in local properties e.g. electrostatic potential or hydrophilic/lipophilic properties, combined with the steric interferences, lead to a loss of specific binding and presumably disables the tumor suppressor function. The 3D-structures combined with molecular biochemical properties of the wild type and the mutated p53-DNA complex can be transferred by the use of the Virtual Reality Modeling Language (VRML). Special tools e.g. ′space buttons′ allow users the interactive exploration of structures, properties, and additional information via internet.

Characterization of specific corticosterone binding sites in adrenal cortex plasma membrane and their localization by autoradiographic studies.

Specific corticosterone binding to calf adrenal cortex plasma membrane was measured using the biologically active radioligand [3H]corticosterone. Corticosterone binding was found to be time-dependent, saturable and reversible, and was reduced by more than 70% when membranes were pretreated with proteases. The population of corticosterone binding sites in calf adrenal cortex plasma membrane was homogeneous and displayed the following characteristics: equilibrium dissociation constant Kd = 77 +/- 8 nM and maximum specific binding capacity Bmax = 70,378 +/- 6,385 fmol/mg protein. The relative affinities of several structural analogues of steroids were deduced from competition assays. From these experiments we can conclude that the plasma membrane binding site characterized is selective for corticosterone and progesterone derivatives, and different from nuclear glucocorticoid, mineralocorticoid, estrogen and progestin receptors. Likewise, this corticosterone binding site is independent of mineralocorticoid and Na+, K(+)-ATPase digitalis receptors. From autoradiographic studies we suggest these corticosterone binding sites are located in the whole adrenal cortex.

Regulation of ferritin expression by oxalomalate in differentiating preadipocytes 3T3-L1

Ferritin content and the specific RNA binding capacity of IRP1/aconitase were determined in lysates of 3T3-L1 adipocytes during their differentiation promoted by the addition to the culture of isobutyl-methylxantine, insulin and dexamethasone (DXM) or by a solution of oxalomalate well known inhibitor of aconitase. The results obtained show that ferritin is present in mature adipocytes and that during differentiation a consistent increase of its formation was observed. Moreover a high extent of RNA-IRPl binding activity was demonstrated only in the DXM differentiated cells, while those treated by presented a binding capacity similar to the control.

Population analysis of the non linear red blood cell partitioning and the concentration‐effect relationship of draflazine following various infusion rates

To investigate the impact of the specific red blood cell binding on the pharmacokinetics and pharmacodynamics of the nucleoside transport inhibitor draflazine after i.v. administration at various infusion rates. It was also aimed to relate the red blood cell (RBC) occupancy of draflazine to the ex vivo measured adenosine breakdown inhibition (ABI). Draflazine was administered to healthy volunteers as a 15-min i.v. infusion of 0.25, 0.5, 1, 1.5 and 2.5 mg immediately followed by an infusion of the same dose over 1 h. Plasma and whole blood concentrations were measured up to 120 h post dose, and were related to the ex vivo measured ABI, serving as a pharmacodynamic endpoint. The capacity-limited specific binding of draflazine to the nucleoside transporter located on the erythrocytes was evaluated by a population approach. The estimate of the population parameter typical value (%CV) of the binding constant Kd and the maximal specific binding capacity (Bmax) was 0.385 (3.5) ng ml-1 plasma and 158 (2.1) ng ml-1 RBC, respectively. The non-specific binding was low. The specific binding to the erythrocytes was a source of non-linearity in the pharmacokinetics of draflazine. The total plasma clearance of draflazine slightly decreased with increasing doses, whereas the total clearance in whole blood increased with increasing doses. The sigmoidal Emax equation was used to relate the plasma and whole blood concentration of draflazine to the ex vivo determined ABI. In plasma, typical values (%CV) of Emax, IC50 and Hill factor were 81.4 (1.9)%, 3.76 (9.3) ng ml-1 and 1.06 (3.4), respectively. The relationship in whole blood was much steeper with population parameter typical values (%CV) of Emax, IC50 and Hill factor of 88.2 (2.0)%, 65.7 (2.8) ng ml-1 and 4.47 (5.5), respectively. The RBC occupancy of draflazine did not coincide with the ex vivo measured ABI. The observed relationship between RBC occupancy and ABI was not directly proportional but similar for all studied infusion schemes. The findings of this study show that the occupancy of the nucleoside transporter by draflazine should be at least 90% in order to inhibit substantially adenosine breakdown in vivo. On the basis of these findings it is suggested that a 15 min infusion of 1 mg draflazine followed by an infusion of 1 mg h-1 could be appropriate in patients undergoing a coronary artery bypass grafting.

Immobilization of microorganisms and proteins on honeycomb monoliths and their composites

Immobilization of biocatalysts on several inorganic monolithic supports has been studied. Transition metals added to TiO2, α-Al2O3 and aluminosilicate change the specific protein binding and support capacity. It is shown that immobilized biocatalysts are stable and can be used to run complex biotechnological processes.

The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions.

The human mitochondrial transcription termination factor (mTERF) cDNA has been cloned and expressed in vitro, and two alternative precursors of the protein have been imported into isolated mitochondria and processed to the mature protein. The precursors contain a mitochondrial targeting sequence, and the mature mTERF (342 residues) exhibits three leucine zippers, of which one is bipartite, and two widely spaced basic domains. The in vitro synthesized mature protein has the expected specific binding capacity for a double-stranded oligonucleotide containing the tridecamer sequence required for directing termination, and produces a DNase I footprint very similar to that produced by the natural protein. However, in contrast to the latter, it lacks transcription termination-promoting activity in an in vitro system, pointing to another component(s) being required for making mTERF termination-competent. A detailed structure-function analysis of the recombinant protein and mutagenized versions of it by band shift assays has demonstrated that both basic domains and the three leucine zipper motifs are necessary for DNA binding. Furthermore, a variety of tests have shown that both the recombinant and the natural mTERF bind to DNA as a monomer, arguing against a dimerization role for the leucine zippers, and rather pointing, together with the results of mutagenesis experiments, to intramolecular leucine zipper interactions being required to bring the two basic domains in close register with the mTERF target DNA sequence.

Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Aims: Mitogenic effect and cellular handling of epidermal growth factor (EGF) were analyzed in primary cultured rat hepatocytes at several cell densities. Methods/Results: DNA synthesis, assessed by the incorporation of 125I-deoxyuridine, was accelerated by EGF at a low cell density while that stimulated by EGF was relatively low at the highest cell density, suggesting a cell density-dependent regulation of mitrogenic response to EGF. An equilibrium binding study of 125-I-EGF in the presence of various concentrations of unlabeled EGF at 0°C revealed that the dissociation constant (K d) was 0.47–0.88 nM while the specific binding capacity (n) was 86–96 fmol/mg protein at each cell density. No significant difference was observed in the time profiles of the surface-bound, internalized, and degradation products of 125I-EGF, assessed per mg protein, between different cell densities. Based on a kinetic analysis of the time-profiles, the internalization rate constant and the degradation rate constant were found to be independent of cell density. Conclusions: These results indicate that the cellular binding and disposition of EGF are not regulated by cell density, and that the cell density-dependence of the mitogenic effect cannot be attributed to differences in the affinity or capacity of the EGF receptor, internalization, or degradation of EGF. We speculate that the cell density-dependent mitogenic response may be accounted for by the difference in other factors such as the signal transduction processes induced by the receptor binding of EGF, or the translocation of a small fraction of the total EGF to hepatocyte nuclei.

CD45-associated protein is a lymphocyte-specific membrane protein expressed in two distinct forms.

CD45-AP is a recently identified CD45-associated protein. The two proteins interact specifically through their respective transmembrane segments. Northern hybridization analysis of CD45+ T lymphocytes and their CD45- variants demonstrated that the production of CD45-AP and CD45 mRNA is regulated independently. On the other hand, Western blotting analysis indicated that the CD45-AP protein has a shorter half-life in the absence of CD45 in three out of four variants. Similar analysis of various types of leukocytes demonstrated that CD45-AP is expressed in T, B, and pre-B cells, but not in plasma cells or cells of the monocyte/macrophage lineage. Two forms of CD45-AP mRNA exist in all types of CD45-AP-expressing lymphocytes analyzed. One form corresponds to the previously reported CD45-AP cDNA and the other form encodes an additional 12 amino acids at the N terminus. The two CD45-AP proteins are identical in their capacity for specific binding to CD45, but employ different mechanisms for endoplasmic reticulum membrane translocation.

Dexamethasone receptors and their distribution in the brain of the red tilapia

Cytosolic extracts of liver and brain showed specific, saturable binding with [3H]dexamethasone: Scatchard plots were linear. Cortisol and 11-deoxycortisol acted as competitors, whilst 17α,20α-dihydroxy-4-pregnen-3-one was relatively much less effective. There was a four-fold range of differences in specific binding capacity between dissected brain regions, the order being telencephalon > hypothalamus > rhombencephalon > optic tectum ≃ (thalamus + tegmentum + torus semicircularis) when Nmax was calculated in terms of DNA content. There was no evidence for any binding of [3;H]dexamethasone with membrane fractions.

Human IgE binding capacity of tryptic peptides from bovine alpha-lactalbumin.

The specific IgE binding capacity of native bovine alpha-lactalbumin (alpha-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow's milk protein allergy. The specific anti-bovine alpha-La IgE titers ranged from 0.6 to 125 IU/ml. Highly purified tryptic peptides from native and disulfide-bond-reduced alpha-La were obtained by reverse phase chromatography. By ELISA technique using immobilized native protein or peptides, 11 of the 19 sera reacted exclusively with intact protein while 8 of them also presented a specific IgE response to different tryptic peptides. Polyclonal IgE population specificity was not related to anti-bovine alpha-La IgE levels. Sequence (17G-K58) and larger peptides sharing this sequence were most strongly and frequently recognized. Competitive ELISA inhibition tests confirmed this IgE-specific response and gave also clear evidence for IgE binding to smaller peptides corresponding to sequences (6C-R10):S-S:(115L-L123) and (109A-L123). IgE binding to native alpha-La and large peptides confirmed the importance of conformational epitope(s). However, in some sera reduced and S-alkylated peptide (59I-K94) exhibited a similar or higher IgE binding capacity than the native corresponding fragment, suggesting the existence of sequential epitope(s) exposed through protein denaturation. Moreover, IgE binding sequences were also located within hydrophobic regions of alpha-La and/or within parts with high sequence homology to human alpha-La.