Immobilized Receptor Research Articles

Covalent immobilization of the estrogen receptor to a cationic N-hydroxysuccinimide ester derivative of agarose

Covalent immobilization of the soluble estrogen receptor from a rabbit uterus to N-hydroxysuccinimide ester derivative of agarose is shown. At first, the condition for the immobilization reaction was examined. The non-immobilized receptor was extracted with 0.4 M NaCl-containing medium. Sixty seven to 80% of the input receptor were immobilized within 30 min at 0°C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4). The immobilized [ 3H]estradiol (3,17β-dihydroxy-1,3,5(10)-estratriene) -receptor complex was stable for at least 24 h. The optimum pH for immobilization was 7.4. Ca 2+ or Na + ions in the reaction media decreased the yields in immobilization of the receptor to the reagent with an electrostatically positive spacer arm. Next, influences of immobilization on the receptor were examined. The dissociation rate of [ 3H]estradiol from the immobilized receptor was a little slower than that from the native receptor. The estrogen-free immobilized receptor was saturated by incubating with 10 nM [ 3H]estradiol for 10 h at 0°C in 0.1 M HEPES (pH 7.4). From Scatchard plot analysis, it was found that the hormone binding affinity in the immobilized receptor decreased to approximately one-fourth of that in the native receptor.

Western blot detection of epidermal growth factor receptor from plasmalemma of culture cells using 125I-labeled epidermal growth factor

We have developed a novel Western blot procedure for the detection of epidermal growth factor (EGF) receptors within a complex mixture of membrane proteins. Purified cell membranes from either human placenta or cultured A431 cells were solubilized, resolved by electrophoresis, and electroblotted onto nitrocellulose paper. With 5–15% gradient gels, electroblotting was completed in 2 h and both the high- and low-molecular-weight proteins were transferred evenly onto the nitrocellulose, as indicated by the radiolabeled protein markers. Upon hybridization with 125I-EGF, the membrane receptor was identified as two adjoining bands on the nitrocellulose of 150 and 170 kDa. Binding of 125I-EGF to the immobilized membrane receptor was specific and was displaced by excess unlabeled EGF. The receptor signal on the autoradiogram was optimized when 1% hemoglobin and 0.05% Tween 20 were present during the hybridization. The ligand-binding activity of the immobilized receptor was not affected by sodium dodecyl sulfate detergent or ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid, but was drastically reduced by either heat denaturation or the addition of dithiothreitol to the membrane samples. Using this method, we were able to demonstrate that no noticeable difference was observed between the pre- and postphosphorylated EGF receptors in their ability to bind to 125I-EGF. Because it allows both identification and purification of a receptor from a mixture of proteins, this protocol should have general application in characterizing various receptor-ligand systems.

PBR322 contains glucocorticoid regulatory element dna consensus sequences

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.

The binding requirements of monkey brain lysosomal enzymes to their immobilised receptor protein

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind \-D-glucosaminidase, α-D-mannosidase, α-L-fucosidase and²-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4.5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an ‘uncovered’ phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.

Kinetic properties of the insulin receptor tyrosine protein kinase: Activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the M r 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 m m ATP, 10 m m Mg (or 10 m m Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity ( K m MnATP ~ 0.01 m m; K m MgATP ~ 0.1 m m). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases V max. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the M r 95,000 (β) subunit; thus the extent of total 32P incorporation into the β subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me 2+ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, M r < 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition. In conclusion, the role of insulin in the activation of receptor protein kinase function is mediately largely (and probably entirely) through the insulin-stimulated, intramolecular, tyrosine-specific autophos-phorylation of the receptor β subunit on a peptide segment of M r < 2000. In turn, autophosphorylation activates the tyrosine protein kinase without altering the affinity of the receptor for the Me 2+ATP or protein substrates and renders the kinase constitutively active and insulin independent.