Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.     

A comparative study of the low-molecular mass serine proteinase inhibitors of human connective tissues.

Low molecular mass serine proteinase inhibitors isolated from human articular cartilage, intervertebral disc, meniscus, and costal cartilage were compared chromatographically. Similar charge and size properties were exhibited when these inhibitors were examined by gel permeation and cation exchange chromatography. The individual proteinase inhibitory species separated by these procedures all cross-reacted with a polyclonal antibody raised against the mucous proteinase inhibitors (MPIs) obtained from human bronchial secretions, however the distribution of these MPI-like species varied with the origin of the connective tissue. The major inhibitory species present in human articular cartilage and intervertebral disc were purified to homogeneity using gel filtration, cation exchange, trypsin affinity and high performance reverse phase chromatography. The amino-terminal sequences of the purified cartilage intervertebral disc inhibitors was found to be identical to the published sequence of MPIs isolated from parotid and seminal secretions. These findings indicate that the endogenous small molecular mass cationic serine proteinase inhibitory proteins present in human cartilaginous connective tissues are members of the MPI family of proteinase inhibitors.     

Comparative analytical studies on recombinant and native human somatotropin

In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin.     

Ferrous ion binding to recombinant human H-chain ferritin. An isothermal titration calorimetry study

Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe(2+) binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe(2+) binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe(2+) travels to the dinuclear ferroxidase center prior to its oxidation to Fe(3+). Calorimetric titrations show that the ferroxidase center is the principal locus for Fe(2+) binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe(2+). That only one site of the ferroxidase center is occupied by Fe(2+) implies that Fe(2+) oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 microM), only 12 Fe(2+)/protein bind at pH 6.51 increasing to 24 Fe(2+)/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe(2+) to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe(2+) binding. The total Fe(2+) binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D131I+E134F), indicating that the primary avenue by which Fe(2+) gains access to the interior of ferritin is through these eight channels. The binding stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe(2+) binding are similar for the two with an average values (DeltaH degrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M(-)(1) at pH 7.04). Since channel mutations do not completely prevent Fe(2+) binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.     

A comparative study of estramustine and pregnenolone binding to prostatic binding protein: evidence for subunit cooperativity

The binding of estramustine, a nitrogen mustard derivative of oestradiol to purified rat prostatic binding protein was studied as a test for a possible identity between this protein and the very similar estramustine-binding protein, described by Forsgren et al. In accordance with this hypothesis estramustine binds to purified prostatic binding protein with a high affinity (2.5 X 10(7)M-1). This affinity markedly exceeds the affinity of pregnenolone for this protein (0.9 X 10(6)M-1) or for a complex of prostatic binding protein, with prostatic proline-rich polypeptide, (4.7 X 10(6)M-1). In competition experiments estramustine completely suppresses the binding of [3H]pregnenolone, whereas the binding of [3H]estramustine is only partially suppressed by pregnenolone, even at high concentrations. Prostatic binding protein was separated in its F- and S-subunit by DEAE-Sepharose chromatography performed in the presence of 8 M urea. Only the S-subunit, most probably in its dimer form, displays marked estramustine and pregnenolone binding, with affinities of respectively 3.7 and 1.2 X 10(6)M-1. Recombination of both subunits results in a strong increase of estramustine binding, but not of pregnenolone binding.     

N-Methyl-D-aspartate receptor subtype-selectivity of homoquinolinate: an electrophysiological and radioligand binding study using both native and recombinant receptors

Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 micro m. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 micro m, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.     

A comparative study of the expression of monoamine oxidase-A and -B mRNA and protein in non-CNS human tissues

The distributions of monoamine oxidase (MAO)-A and -B proteins and mRNAs in human heart, lung, liver, duodenum, kidney and vasculature were compared using immunohistochemistry and cRNA in situ hybridisation. MAO-A and -B mRNA were detected in all tissues, to differing extents, but particularly in glomeruli, hepatocytes, and the crypts, muscularis mucosa and muscularis externa of duodenum. Renal proximal and distal tubules and loops of Henle had more intense labelling for mRNA of MAO-B than MAO-A; this was reflected in MAO protein expression. Little immunoreactivity was detected in glomeruli. Hepatocytes expressed MAO-A moderately, but MAO-B strongly. In lungs, similar moderately intense labelling for both MAO mRNAs and immunoreactivities was evident in pneumocytes, and epithelial and smooth muscle cells. Cardiomyocytes contained both MAO isoforms, but with more, albeit moderate, labelling for MAO-A. Both isoforms were expressed equally in duodenal villi, crypts, muscularis externa and mucosa; lower level expression occurred in mucosal and submucosal cells. MAO-A and -B mRNA were detected in endothelia, adventitia and media of a renal interlobular artery, but protein immunoreactivities were chiefly in the adventitia. The data reveal widespread tissue distribution of MAO mRNAs and proteins, but indicate that presence of MAO mRNAs does not invariably reflect quantitatively its protein expression.     

Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase

Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady-state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ.mol-1 and 12.34 kJ.mol-1, in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ x mol(-1) and 12.34 kJ x mol(-1), in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

A lectin histochemistry comparative study in human normal prostate, benign prostatic hyperplasia, and prostatic carcinoma

The partial oligosaccharide sequences of glycoconjugates and the nature of their glycosidic linkages were investigated in normal human prostate, benign prostatic hyperplasia (BPH) and prostatic carcinoma by means of lectin histochemistry, using light microscopy and Western blot analysis. The labeling pattern of BPH differed from that of normal prostate in having more intense staining with DSA, HPA, UEA-I and AAA, and in showing lesser staining with WGA and SBA. Prostatic carcinoma differed from normal prostates in displaying the more intense labeling with PNA, DSA, SBA, DBA, UEA-I and AAA, and in having lesser labeling with WGA. The main differences in labeling pattern between prostatic carcinoma and BPH were that the latter specimens showed more marked staining with PNA, DSA, DBA, SBA, UEA-I and AAA, and lesser staining with WGA and HPA. The staining patterns of SNA, MAA, ConA, LCA and GNA were similar in all three groups of specimens. For most of the lectins studied, including those showing a similar immunohistochemical staining in the three groups of specimens studied, the Western blot analysis showed differences in the banding pattern among normal, hyperplastic, and carcinomatous prostates. Present results suggest that the glycosylation of proteins was modified in both BPH and prostatic carcinoma. In BPH a strong expression of N-acetylgalactosamine residues occurred, while in prostatic carcinoma an increase of sialic aci, galactose and fucose residues was observed. No changes in mannose residues were detected.     

Hydrodynamic and spectroscopic studies of substrate binding to human recombinant deoxycytidine kinase

Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.     

A comparative study of surface binding of human low density and high density lipoproteins to human fibroblasts: regulation by sterols and susceptibility to proteolytic digestion.

Binding of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein (HDL) was determined in cultured human fibroblasts from a normal subject and two subjects with homozygous familial hypercholesterolemia (HFH). Binding was assayed at 0 degree C to minimize the internalization of labeled lipoproteins. The binding of LDL and of HDL were compared following interventions reported to affect LDL binding in normal fibroblast. LDL binding to normal cells increased two to three fold 24 hours after transfer from medium containing whole fetal calf serum to medium containing lipoprotein-deficient fetal calf serum. This increase was completely blocked in the presence of cycloheximide (200 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). This increased capacity of normal fibroblasts to bind LDL could be reduced 70-80% by a subsequent 18-hour incubation with cholesterol (50 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). In contrast, no significant change in HDL binding to normal fibroblasts was observed after any of these interventions. HFH cells to show any significant change in either LDL binding or HDL binding following these interventions. These results suggest that HDL binding sites on normal fibroblasts are for the most part distinct from LDL binding sites. They also support the conclusion that LDL binding sites on HFH cells are for the most part qualitatively different from those on normal cells.