Biospecific labeling with undecagold: visualization of the biotin-binding site on avidin.

The biotin-binding site on avidin has been labeled with biotin conjugated to undecagold, an organometallic cluster compound containing 11 gold atoms in a core angestroms in diameter. Examination of unstained specimens by scanning transmission electron microscopy reveals the labeled sites directly, without computational averaging or filtering of the images. This approach should be widely applicable for determining the locations of subunits and functional site in biological macromolecules at a resolution at a resolution in range of 15 angstroms.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Formation of complexes between avidin and beta-adrenergic receptors using biotinyl-alprenolol derivatives.

The goal of this study was to synthesize biotinylated derivatives of alprenolol, a beta-adrenergic antagonist, and to determine whether these ligands could bind simultaneously to both avidin (a biotin-binding protein) and to the beta-adrenergic receptor. Such ligands would be useful for beta-adrenergic receptor localization and purification, since avidin can be covalently labelled with fluorescent or electron-dense markers or can be linked to solid supports for affinity chromatography. Three biotinyl derivatives of alprenolol were synthesized and characterized. Each derivative bound to avidin and also possessed high affinity for the duck erythrocyte beta-adrenergic receptor. Two of the compounds, biotinyl-caproyl-cysteaminyl-alprenolol (BCCA) and biotinyl-dodecanoyl-cysteaminyl-alprenolol (BDCA) had the same affinities for the duck erythrocyte beta-adrenergic receptor (membrane-bound or digitonin-solubilized) in the absence and presence of avidin. This indicated that high affinity complexes could be formed between the beta-adrenergic receptor and avidin using these bifunctional biotinyl-alprenolol ligands. In contrast, biotinyl-cysteaminyl-alprenolol (BCA), in which the distance between the biotin and alprenolol moieties was shorter, had greatly reduced affinity for the duck erythrocyte beta-adrenergic receptor in the presence of avidin. Additional studies showed that BDCA, avidin-BDCA, and ferritin-avidin-BDCA were equally potent in inhibiting the isoproterenol stimulation of cAMP accumulation in intact HeLa cells. The data reported in this paper demonstrate the importance of an appropriate spacer sequence to allow correct apposition of the receptor and avidin molecules, and suggest that BDCA may be a useful probe for beta-adrenergic receptor localization and purification.

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography.

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability.

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.

Purification of biotin-binding protein from chicken egg yolk and comparison with avidin.

A simple alternative procedure for the purification in higher yields of the biotin-binding protein from the chicken egg yolk in a ligand-free form is described. The isolated protein was homogeneous by the criteria of polyacrylamide gel electrophoresis, gel filtration chromatography, immuno-double diffusion and immuno-electrophoresis. The protein had an Mr of 72 000 +/- 2000 and was a homotetramer of subunit Mr of 18 000 +/- 1000. It bound [14C]biotin in the molar ratio of 1:4 with an association constant (Ka) of 0.58 X 10(12) M-1. The yolk biotin-binding protein and avidin exhibited qualitatively similar spectral changes on interaction with biotin and p- hydroxyazobenzoic acid, but quantitatively these changes were more pronounced with avidin. Despite the lack of gross immunological cross-reactivity between the two biotin-binders, evidence based on immunological techniques for some degree of common conformational characteristics restricted to or around the ligand-binding sites of the two proteins was adduced. The mixed subunits of the two proteins failed to form hetero-oligomers on reconstitution.

Avidin binding of biotinylated analogues of substance P.

We report the bioactivities of three biotinylated analogues of Substance P, [alpha-biotinyl-Lys3]Substance P-(3-11), [alpha-biotinyl-Arg1]Substance P, [epsilon-biotinyl-Lys3]Substance P, as well as the bioactivities of their complexes with avidin on guinea pig ileum. The rate of dissociation of an [alpha-biotinyl-Arg1]Substance P-avidin complex has been determined in the presence of 2-(4'-hydroxyazobenzene) benzoic acid and [3H]biotin. The biphasic dissociation of a 4:4 complex between [alpha-biotinyl-Arg1]Substance P and avidin led us to postulate the existence of two sets of binding sites, with the following rates of dissociation, at 4 degrees C, kappa-1 = 6 x 10(-4) x s-1 and kappa-1 = 1.2 x 10(-5) x s-1. We have confirmed this non-equivalence of the four binding sites of avidin by nuclear magnetic resonance using a spin-echo technique. The [alpha-biotinyl-Arg1]Substance P-avidin may be used for the affinity chromatography of Substance P receptors and the decreased affinity of this complex may be taken as an advantage since it can be displaced under mild conditions, i.e. by biotin-containing buffer.

Similarity of biotin-binding activity and immunoreactivity in chicken oviduct and non-oviduct avidin.

Biotin-binding and immunological methods were employed to demonstrate the similarity of oviduct and non-oviduct avidin in the chicken. Oviduct avidin was induced after oestrogen pretreatment by progesterone and non-oviduct avidin by intestinal tissue injury or by intraperitoneal actinomycin D administration. Avidin in the intestine, lung, bursa of Fabricius, plasma, pectoral muscle and liver after injury had biotin-binding activity similar to that of progesterone-induced oviduct avidin: (1) a temperature of 79-83 degree C was required for 50% of the maximum [14C]biotin uptake, (2) maximal exchange occurred only at 90 or 100 degree C and (3) denaturation of protein, i.e., loss of biotin-binding activity, was not yet observed at 100 degree C. Avidin in the intestine, lung, bursa of Fabricius, plasma and pectoral muscle also showed an identical cross-reaction with oviduct avidin. Furthermore, the increase in avidin-like biotin binding in the oviduct and most non-oviduct tissues was significantly correlated with the increase in avidin-like antigen in the tissue. This indicates that avidin induced in chicken non-oviduct tissues by injury or inflammation caused by actinomycin D administration is similar to progesterone-dependent oviduct avidin.

The characterization of the uptake of avidin-biotin complex by HeLa cells.

HeLa cells have been shown to internalize the avidin-biotin complex. Adsorptive pinocytosis seems to be the mechanism of this uptake as seen by the requirements of energy and the integrity of the microtubular assembly. Pretreatment of HeLa cells with cycloheximide inhibits uptake and binding of the avidin-biotin complex. Scatchard plot of specific binding of avidin indicates a single type of binding with a Kd of approx. 55 pM with about 21,500 receptors/cell. The lack of inhibition of binding by simple carbohydrates indicates that binding is not through the oligosaccharide chain of avidin.

Selective binding of fluoresceinated-avidin to A-cells of sectioned pancreas.

Fluoresceinated-avidin (FITC-avidin) was observed to bind specifically to a subset of pancreatic islet cells in sections of both human and rat pancreas. FITC-avidin binding was inhibited by excess unlabeled avidin, and by biotin, but not by glucagon, somatostatin, or insulin. Labeling of islets with anti-insulin, anti-glucagon, anti-somatostatin, and anti-human pancreatic polypeptide antibodies showed the avidin binding subset to correspond to islet cells identified by anti-glucagon antibody. Conversely, avidin reacted with no insulin, somatostatin, or cells containing HPP. We conclude that avidin localizes specifically to A-islet cells. Binding may be to a biotin-containing enzyme within the A-cells, but the precise molecular site of binding is currently unidentified.

Crystallization of hen egg-white avidin in a tetragonal form.

Hen egg-white avidin has been crystallized at pH 5.7 from ammonium sulfate solutions. The crystals belong to the tetragonal space group P4(2)2(1)2, with unit cell edges a = b = 79.6 A, c = 84.3 A. Assuming a molecular weight of 15,600 per avidin monomer, this crystal form is compatible with the presence of a dimer in the asymmetric unit, and is suitable for a crystallographic structural investigation at high resolution.

Immunogenicity of biotinylated hapten-avidin complexes.

The efficacy of avidin as a carrier for the generation of anti-hapten antibodies was assessed in mice by immunization with complexes of avidin and synthetic peptides containing biotin and an epsilon-dinitrophenyl (DNP) lysine residue. The synthetic haptens were constructed with 0, 1 or 2 6-aminocaproyl groups as spacers between the biotin and DNP-lysine moieties. Complexes without a spacer did not induce anti-DNP antibody responses, while those with two spacers induced stronger responses than those with only one spacer. However, the anti-DNP responses to avidin-biotinylated hapten complexes were considerably weaker than responses to a conventional hapten-protein conjugate (DNP-ovalbumin), and, like "T-independent" antigens, failed to induce significant immunological memory. The distribution of isotypes in the anti-DNP antibodies produced to avidin-biotin-6-aminocaproyl-epsilon-DNP-lysine-alanine and DNP-ovalbumin was similar, but the former antigen induced significantly lower levels of antibody in (CBA/N X BALB/c) F1 male mice with the xid defect than in phenotypically normal female littermates, and also induced significant responses in nu/nu mice, in contrast to DNP-ovalbumin. These findings suggest that there is at least a "T-independent" or "T-efficient" component in the response to avidin-biotin complexes, perhaps due to the tetrameric structure of the molecule. Estimates of the depth of the receptor site for biotin were obtained by using the complexes to competitively inhibit the binding of anti-DNP antibody to plates coated with DNP-protein. The findings were consonant with the data on immunogenicity (capacity to induce anti-DNP antibody responses) and suggested that the receptor site has a depth of 16-26 A.

Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods.

Indirect immunofluorescence techniques for labeling cell surface antigens on murine pluripotent hemopoietic stem cells often result in a reduction of CFU-S numbers. This phenomenon was investigated by comparing indirect immunofluorescence and biotin-avidin methods using anti-T200 and anti-H-2Kk monoclonal antibodies. Mouse bone marrow cells treated with these monoclonal antibodies, alone or in combination with fluorescein conjugates of rabbit antirat or goat antimouse immunoglobulins, respectively, showed reduced numbers of CFU-S. The reduction in CFU-S numbers by anti-H-2Kk antibodies was dependent on the concentration of antibody and on the antigen density on the cells. Near complete CFU-S recovery was obtained with biotin-labeled antibodies and avidin-fluorescein isothiocyanate. The CFU-S recovery obtained was higher with higher numbers of biotin moieties per antibody molecule. Biotinylation itself did not influence the antibody binding properties. The protective effect was independent of the avidin-FITC concentration. Injection of carrageenan, an agent known to block macrophage activity, prevents the reduction of CFU-S recovery caused by anti-H-2Kk antibody treatment. The biotin-avidin procedure permits the measurement of antigen density on pluripotent stem cells through flow cytometry and sorting and full recovery of CFU-S in the in vivo assay.

Chicken macrophages synthesize and secrete avidin in culture.

It was previously shown that avidin, a glycoprotein secreted in vivo by chicken oviduct, is produced by cultured transformed or damaged chicken embryo fibroblasts [27]. This report demonstrates synthesis and secretion of large amounts of avidin by macrophages isolated from chicken yolk sac. Avidin was secreted to the culture medium as shown by immunoprecipitation of metabolically labeled proteins. In the culture medium of macrophages the avidin concentration (up to 47.5 +/- 0.5 microgram/mg cellular protein) exceeded, in agreement with previous findings, that of fibroblasts (up to 7.3 +/- 0.7 microgram/mg) infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature sensitive for transformation and OK 10 virus). No difference between the macrophage avidin and the egg white avidin was detected by both the heat-induced [14C] biotin exchange assay and immunoblotting (subunit Mr = 15600). By immunofluorescence 10 to 20% of the cells were positive for avidin, independent of the time in culture (1-30 days). The staining pattern varied between dense or granular perinuclear and strong reticulo-granular fluorescence throughout the cytoplasm. Double staining for avidin and the Golgi region by wheat germ agglutinin showed that avidin is concentrated, and might be processed, in the Golgi complex. The production of avidin by macrophages supports a role for avidin in host defence mechanisms.

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique.

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.

Conjugated avidin identifies cutaneous rodent and human mast cells.

Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first with conjugated avidin, and then with a metachromatic stain demonstrated that both techniques identify the same mast cell granules. Specificity for mast cells was confirmed by the absence of avidin-positive cells in the skin of mast cell-deficient (W/Wv) mice. Binding of conjugated avidin to mast cells was inhibited by pretreating tissue specimens with unconjugated avidin but not by pretreating conjugated avidin with biotin, indicating that avidin does not bind to biotin or a biotin-like molecule. Within murine dermis, unique patterns of mast cell distributions were observed, with a prominent perivascular localization in ear skin, and a complete absence of mast cells underlying the scales in tail skin. In tissue sections of guinea pig skin undergoing basophil hypersensitivity reactions and in murine and human skin specimens infiltrated with eosinophils, conjugated avidin selectively stained only dermal mast cells, demonstrating specificity for mast cells in sites of inflammation. Conjugated avidin also readily stained rat peritoneal mast cells, demonstrating its utility for identifying extracutaneous mast cells. Unlike the metachromatic stains, avidin binding to mast cells in tissues is not limited by methods of fixation or special embedding and cutting procedures. Thus, mast cell identification with conjugated avidin is a reliable, specific, and simple method with important clinical and investigative applications.

Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct.

The messenger RNA (mRNA) for avidin, which represents less than 0.05% of the total cellular proteins, was partially purified from hen oviduct, and the presence of avidin mRNA was shown to depend upon prior stimulation by progesterone. A total nucleic acid extract was subjected to oligo (dT)-cellulose chromatography, followed by Sepharose 4B chromatography, preparative agarose gel electrophoresis, and sucrose gradient centrifugation. The relative purity of each preparation was assessed by translation in a wheat-germ system; avidin messenger RNA activity was measured by specific immunoprecipitation of synthesized proteins. Avidin mRNA was separated from the bulk of the total messenger RNA activity of the oviduct and from all ribosomal RNAs to produce greater than a 1000-fold enrichment of avidin mRNA activity compared with total cellular RNA. Based on the translation assay, the most highly purified fraction contained about 2.5% avidin messenger RNA. Avidin mRNA activity was absent in partially purified mRNA obtained from estrogen-stimulated chick oviducts, but was detected in oviducts following progesterone administration.

Progesterone-independent avidin in chick oviduct fibroblast culture.

The production of avidin was studied in chick oviduct cell cultures derived from immature chicks or from chicks with 4, 8, or 14 days of estrogen priming in vivo. Cells were grown for 5--7 weeks, and the monolayers formed were composed of collagen-producing fibroblasts. In some cultures, epithelial cells were also found, but only in the original explants. Two-day avidin production of cultures was measured in the media weekly. Cultures produced avidin spontaneously, the amount being fairly stable during the 7-week culture period. No difference was found in avidin production or cell morphology when estrogen-containing medium was used. Cultures from 4- to 8-day-estrogen-primed chick oviducts produced the same amount of avidin as cultures from immature oviducts, whereas further estrogen pretreatment seemed to reduce avidin production. Progesterone did not enhance avidin production with or without estrogen priming but, due to its inhibition of growth, clearly inhibited avidin when it was continuously in the culture medium. It is concluded that chick oviductal fibroblasts have an inherent capacity for avidin production and that this is independent of progesterone.

Estrogen regulation of the biological activity of the avian oviduct progesterone receptor and its ability to induce avidin.

During primary estrogen stimulation of chick oviduct development, estrogen withdrawal, or secondary estrogen treatment, changes in the oviduct progesterone receptor (PR) occur. The presence of estrogen appears to regulate not only PR concentration but also its biochemical activity, i.e. its capacity to bind to nuclear acceptor sites and alter RNA synthesis. This study reports that estrogen regulates the nuclear binding capacity of the PR even more rapidly than previously reported in fully developed oviducts of chicks that have been injected daily for 4 weeks with diethylstilbestrol (DES). Further, the nuclear binding capacity of the PR correlates with the ability of progesterone (P) to induce avidin protein concentrations in the oviducts in vivo. The PR concentration in the oviducts increases 2-fold within 8 h of the last injection and the decreases to a minimal value by 24 h. Injection of [3H]P into the chicks shows that the in vivo nuclear localization of the steroid increases almost 4-fold at 8 h, followed by a similar decrease to minimal values by 24 h. Cell-free nuclear binding assays, using PR isolated at various times after the last DES injection and oviduct nucleoprotein complexes, indicate that the capacity of the receptors to bind to nuclear acceptor sites is regulated by the estrogen. The enhanced nuclear binding capacity of the isolated PR increases to maximal values by 12-14 h after the last estrogen treatment and then begins to decrease to minimal values by 24 h. Similarly, the ability of P to induce in vivo avidin protein concentrations and to alter general RNA synthesis in the oviducts is reduced by 70% (of the estrogen non-withdrawn chick levels) by 24 h after the last estrogen injection. These changes over the 24-h period after the last DES treatment are not due to changes in the serum DES concentrations. The following 10-day period of estrogen withdrawal reveals a cyclic decaying pattern in the capacity of the PR for nuclear binding. The P induction of avidin and alteration of RNA polymerase II activity, using nuclear run-off experiments, also show a similar cyclic decaying pattern. By 6 days of estrogen withdrawal, the PR is incapable of any nuclear binding, and P cannot induce avidin protein concentrations in the oviducts. Serum DES concentrations over this 10-day period display only a gradual decay.(ABSTRACT TRUNCATED AT 400 WORDS)

Avidin induction by theophylline in vivo and in vitro.

Avidin induction in chick tissues in vivo and in vitro was studied by a phosphodiesterase inhibitor, theophylline, and compared to progesterone-dependent induction. Theophylline (100 mg/kg, ip) caused a significant increase in avidin content only in the oviduct of diethylstilbestrol-treated chicks, but not in the lung, muscle, intestine, plasma, or in the bursa of Fabricius. Diethylstilbestrol priming was necessary for oviductal avidin induction in vivo by theophylline. In the oviduct culture, theophylline at a concentration between 100 and 500 micrograms/ml caused a dose-dependent increase in avidin production. Effects of theophylline and progesterone on avidin synthesis in oviduct culture were synergistic. Avidin production was dependent on protein and RNA synthesis, since induction was inhibited by cycloheximide and actinomycin D. Avidin induction by theophylline resembled progesterone-dependent induction, beginning 9 h after the injection in vivo and 12 h after administration of these drugs in vitro. Avidin induced by theophylline showed heat-induced biotin exchange identical to that of progesterone-induced avidin, indicating close similarity of these proteins. The results suggest that theophylline can mimic the action of progesterone on avidin production, and that cyclic nucleotides may have a role in the regulation of avidin synthesis.