Presence Of Binding Protein Research Articles

Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology.

According to the free hormone hypothesis, biological activity of a certain hormone is best reflected by free rather than total hormone concentrations. A crucial element in this theory is the presence of binding proteins, which function as gatekeepers for steroid action. For testosterone, tissue exposure is governed by a delicate equilibrium between free and total testosterone which is determined through interaction with the binding proteins sex hormone-binding globulin and albumin. Ageing, genetics and various pathological conditions influence this equilibrium, hereby possibly modulating hormonal exposure to the target tissues. Despite ongoing controversy on the subject, strong evidence from recent in vitro, in vivo and human experiments emphasizes the relevance of free testosterone. Currently, however, clinical possibilities for free hormone diagnostics are limited. Direct immunoassays are inaccurate, while gold standard liquid chromatography with tandem mass spectrometry (LC-MS/MS) coupled equilibrium dialysis is not available for clinical routine. Calculation models for free testosterone, despite intrinsic limitations, provide a suitable alternative, of which the Vermeulen calculator is currently the preferred method. Calculated free testosterone is indeed associated with bone health, frailty and other clinical endpoints. Moreover, the added value of free testosterone in the clinical diagnosis of male hypogonadism is clearly evident. In suspected hypogonadal men in whom borderline low total testosterone and/or altered sex hormone-binding globulin levels are detected, the determination of free testosterone avoids under- and overdiagnosis, facilitating adequate prescription of hormonal replacement therapy. As such, free testosterone should be integrated as a standard biochemical parameter, on top of total testosterone, in the diagnostic workflow of male hypogonadism.

Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non-sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol-binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti-inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment.

Altered mechano-chemical environment in hip articular cartilage: effect of obesity

The production of extracellular matrix (ECM) components of articular cartilage is regulated, among other factors, by an intercellular signaling mechanism mediated by the interaction of cell surface receptors (CSR) with insulin-like growth factor-1 (IGF-1). In ECM, the presence of binding proteins (IGFBP) hinders IGF-1 delivery to CSR. It has been reported that levels of IGF-1 and IGFBP in obese population are, respectively, lower and higher than those found in normal population. In this study, an experimental-numerical approach was adopted to quantify the effect of this metabolic alteration found in obese population on the homeostasis of femoral hip cartilage. A new computational model, based on the mechano-electrochemical mixture theory, was developed to describe competitive binding kinetics of IGF-1 with IGFBP and CSR, and associated glycosaminoglycan (GAG) biosynthesis. Moreover, a gait analysis was carried out on obese and normal subjects to experimentally characterize mechanical loads on hip cartilage during walking. This information was deployed into the model to account for effects of physiologically relevant tissue deformation on GAG production in ECM. Numerical simulations were performed to compare GAG biosynthesis in femoral hip cartilage of normal and obese subjects. Results indicated that the lower ratio of IGF-1 to IGFBP found in obese population reduces cartilage GAG concentration up to 18% when compared to normal population. Moreover, moderate physical activity, such as walking, has a modest beneficial effect on GAG production. The findings of this study suggest that IGF-1/IGFBP metabolic unbalance should be accounted for when considering the association of obesity with hip osteoarthritis.

Abstract 4477: Progesterone (P4) and testosterone (T) concentrations in random fine needle aspirates of the breast in pre- and postmenopausal women

Abstract Background: The availability of steroid hormones in the breast may be an important measure of the potential for proliferation of hormone-dependent breast cancer, and because retention of hormones may be influenced by serum levels, in situ formation, and the presence of binding proteins and lipids in the tissue, measurements of tissue concentrations are more informative than serum concentrations alone. Methods: Subjects were normal women, aged 36-65 yr, without breast cancer and not taking hormones. Tissue was obtained by fine needle aspiration of the breast. The cellular pellet was saved for analysis of DNA, and lipids were extracted from the supernatant including the fatty layer. Previous work had shown the cellular pellet contained a very little of the total steroids of the sample. Triglycerides were precipitated from 90% methanol at –20 °C and the remaining lipids were applied to a C18 HPLC column for purification in a gradient of methanol and acetonitrile in phosphate buffer. Fractions containing P4 and T were analyzed by immunoassays. The total DNA in the cellular pellet was isolated and quantified by UV absorbance. The steroid content was expressed as either per mg of purified lipid or per microgram of DNA. The data, when expressed as the natural log of the concentrations, followed a normal distribution. Differences in pre- and postmenopausal concentrations were analyzed by group t-tests. Results: The geometric means of the hormone concentrations are shown in the table. Comment: Despite the almost complete loss of progesterone in serum of postmenopausal women, the concentrations in the tissue are reduced but remain, on average, at more than 25% of the premenopausal level, somewhat more than the previously reported percentage in NAF of 5%. Relationships within individual women to other risk factors will be explored. Testosterone is also maintained at a relatively high level in the tissue of postmenopausal women, and may serve as a substrate for aromatase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4477. doi:1538-7445.AM2012-4477

Antibodies to the endoplasmic reticulum-resident chaperones calnexin, BiP and Grp94 in patients with rheumatoid arthritis and systemic lupus erythematosus

To investigate the presence of autoantibodies against mammalian chaperones of the endoplasmic reticulum (ER) in patients with RA and other immune-mediated diseases. Sera from healthy donors, from early RA patients with two follow-up samples, patients with SLE, SSc and IBD were collected and analysed for anti-ER chaperone antibodies. Detection of serum IgG antibodies against immunoglobulin heavy chain binding protein (BiP), glucose-regulated protein 94 (Grp94) and calnexin was carried out using ELISA. The specificity of sera positive for individual ER chaperones was confirmed by immunoblotting. Statistical analysis was performed using Welch's t-test, Mann-Whitney U-test, partial correlation and Pearson's correlation. In patients with RA and SLE, autoantibody titres against BiP, Grp94 and calnexin were significantly higher than those in healthy controls. These autoantibodies were detectable in patients with early RA and titres remained stable for at least 6-12 months. Also several SSc and IBD patients exhibited autoantibodies against these ER chaperones; however, titres and frequencies were lower than in RA or SLE patients. Furthermore, anti-calnexin antibodies correlated significantly with the presence of BiP and Grp94 autoantibodies in patients with RA and SLE. Calnexin and Grp94 were identified as novel autoantigens in RA and calnexin in SLE. Since calnexin, Grp94 and BiP are ER-resident proteins of eukaryotic cells, our data suggest that autoantibody generation against ER chaperones is independent of initial exposure to the corresponding bacterial chaperones; rather, ER chaperones may represent genuine autoantigens.

Co-expression of 9-O-acetylated sialoglycoproteins and their binding proteins on lymphoblasts of childhood acute lymphoblastic leukemia: an anti-apoptotic role

Enhanced levels of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)GPs) as disease-associated molecules was reported to act as signaling molecules for promoting survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Here, we searched for potential physiological ligands for Neu5,9Ac(2)GPs that could be involved in modulating the survival of lymphoblasts. Accordingly, we examined the presence of binding proteins for Neu5,9Ac(2)GPs on cell lines and primary cells of patients with B- and T-ALL, at presentation of the disease. Peripheral blood mononuclear cells from normal healthy donors and cells from myeloid leukemia patients were used for comparison. Neu5,9Ac(2)GPs-binding proteins (BPs) were specifically detected on the surface of both T- and B-ALL-lymphoblasts and ALL-cell lines along with the consistent presence of Neu5,9Ac(2)GPs. The Neu5,9Ac(2)GPs and BPs also co-localized on the cell surface and interacted specifically in vitro. Apoptosis of lymphoblasts, induced by serum starvation, was reversed in the presence of purified Neu5,9Ac(2)GPs due to possible engagement of BPs, and the anti-apoptotic role of this interaction was established. This is the first report of the presence of potential physiological ligands for disease-associated molecules like Neu5,9Ac(2)GPs, the interaction of which is able to trigger an anti-apoptotic signal conferring a survival advantage to leukemic cells in childhood ALL.

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98–106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.

Sensing DNA–DNA as Nanosensor: A Perspective Towards Nanobiotechnology

(Dated: 2nd February 2008)Based on modern single molecule techniques, we devise a number of possible experimental setupsto probe local properties of DNA such as the presence of DNA-knots, loops or folds, or to obtaininformation on the DNA-sequence. Similarly, DNA may be used as a local sensor. Employingsingle molecule fluorescence methods, we propose to make use of the physics of DNA denaturationnanoregions to find out about the solvent conditions such as ionic strength, presence of bindingproteins, etc. By measuring dynamical quantities in particular, rather sensitive nanoprobes may beconstructed with contemporary instruments.Key words: DNA, DNA breathing, single molecule spectroscopy, nanosensors, fluorescence correla-tion spectroscopy, fluorescent resonance energy transfer

Inhibition of antigen-presenting cell function and stimulation of human peripheral blood mononuclear cells to express an antiinflammatory cytokine profile by the stress protein BiP: relevance to the treatment of inflammatory arthritis.

The stress protein and endoplasmic reticulum chaperone, immunoglobulin binding protein (BiP), is an autoantigen in rheumatoid arthritis (RA). Stress proteins, however, may have extracellular functions, mediated via cell surface receptors, that may include immunomodulatory functions. We sought to determine whether cell-free BiP is present in the synovial fluid (SF) of patients with RA and to further investigate the possible extracellular antiinflammatory and immunomodulatory properties of BiP in peripheral blood mononuclear cells (PBMCs) in vitro. The presence of BiP in SF was established by Western blotting. PBMCs were stimulated with exogenous recombinant human BiP, and cytokine production and cell proliferation were measured in the presence and absence of cell signaling inhibitors or neutralizing anti-interleukin-10 (anti-IL-10) monoclonal antibody. Cytokine levels were quantified by enzyme-linked immunosorbent assay, cell proliferation by tritiated thymidine uptake, and cell surface molecule expression by flow cytometry. PBMCs responded to BiP with secretion of an antiinflammatory profile of cytokines. Although BiP stimulated the early production of tumor necrosis factor alpha (TNF alpha), the major cytokine induced was IL-10. Soluble TNF receptor II and IL-1 receptor antagonist secretion was also increased. Addition of SB203580, the MAPK p38 pathway inhibitor, partially inhibited the production of IL-10 and TNF alpha, whereas they were unaffected by the MAPK ERK-1/2 inhibitor PD98059. BiP also inhibited the recall antigen response by PBMCs to tuberculin purified protein derivative. Further investigation showed that incubation of monocytes in the presence of either BiP or IL-10 down-regulated CD86 and HLA-DR expression. The effect observed with IL-10 was transient compared with the long-lasting reduction induced by BiP. Extracellular BiP may stimulate immunomodulatory and antiinflammatory pathways, which are only partly due to the production of IL-10. These properties may be of relevance for the treatment of diseases such as RA.

Regulation of isomerohydrolase activity in the visual cycle.

While the overall biosynthetic pathway leading from all-trans-retinoids to 11-cis-retinoids in the visual cycle is understood, little is known about which step(s) may be rate-limiting and how control is exerted. One possible target for control is the isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-retinol. The basal rate of 11-cis-retinol synthesis from all-trans-retinyl esters is extremely slow using bovine retinal pigment epithelial membranes [3.5 pmol of 11-cis-retinol min-1 (mg of protein)-1], and only small amounts of 11-cis-retinyl ester are formed. However, the addition of retinol binding proteins stimulates 11-cis-retinol formation by a factor of approximately 13. Specific protein-protein interactions are probably unimportant because bovine serum albumin and the physiologically relevant cellular retinaldehyde binding protein (CRALBP) both stimulate 11-cis-retinol formation to the same extent, although CRALBP does so at much lower concentrations. The relatively rapid rate of isomerization in the presence of binding proteins [44.3 pmol of 11-cis-retinol min-1 (mg of protein)-1] suggests that the rate-limiting enzyme in the visual cycle need not be the isomerohydrolase. Also, 11-cis-retinol is shown to inhibit isomerohydrolase, providing a simple mechanism for regulation of the visual cycle and the stimulating effect of binding proteins.

Is there facilitated uptake of fatty acids by the liver? Interpretation and analysis of experimental data.

Uptake of hydrophobic organic anions that are extensively bound to serum proteins has been a controversial issue for over 30 years. It is known that steady-state uptake is lower in the presence of binding proteins, but it is much higher than predicted on the basis of protein-ligand binding equilibrium. Several theories have been postulated to account for this observation. Recent work has shown how binding proteins are capable of enhancing the uptake rate of long-chain fatty acids by decreasing the diffusional resistance of the unstirred fluid layer. The enhanced transport via codiffusion is especially important for tightly bound ligands like long-chain fatty acids. Whether this model accounts for all experimental data or whether hepatocytes facilitate the uptake of protein-bound ligands, by for example mediating the protein-ligand dissociation rate, is not clear. We review the published reports to gain an understanding into the potential mechanism for the extraction of long-chain fatty acids. Understanding the uptake mechanism of these important metabolic substrates is vitally important in determining their overall utilization in a variety of clinical disorders as diverse as gallstones, obesity, and atherosclerosis.

Pharmacokinetics and pharmacodynamics of recombinant proteins and peptides

Recombinant derived proteins and peptides are becoming increasingly important as therapeutic agents. The successful development of recombinant proteins depends on the proper characterization of their pharmacokinetics and an understanding of the relationship between drug exposure or dose and the pharmacological response. Although many traditional pharmacokinetic principles can be applied to recombinant proteins and peptides, biotechnologically derived agents also pose some new challenges. Pharmacokinetic characterization of proteins is often complicated due to the absence of a specific assay, difficulties in identifying metabolites, endogenous circulating concentrations, and the presence of binding proteins. The ubiquitous distribution of proteases leads to significant degradation and relatively short circulation times in the body. The protein concentration in the circulation may not correlate with the pharmacological effect and it may be difficult to identify or sample the biophase. Numerous methods have been explored for increasing the residence time of proteins and enhancing the pharmacological effect. Some of the techniques that have been employed include site-specific mutagenesis, polymer-modifications and fusion with immunoglobulins, targeting through the use of liposomes or conjugation to toxins, radionuclides and other proteins. An increase in the residence time does not always lead to an enhanced therapeutic effect and these methods have resulted in variable degrees of success. The intricate relationship between pharmacokinetic and pharmacodynamic properties of recombinant proteins is discussed. Examples are presented to illustrate the diversity of approaches that have been employed to improve the therapeutic efficacy of recombinant proteins.

1,25-Dihydroxyvitamin D3 in the Atlantic cod: plasma levels, a plasma binding component, and organ distribution of a high affinity receptor.

Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.

Paracrine regulation of reproductive function by inhibin and activin.

ConclusionsInhibin has been confirmed as a factor produced in the gonads and capable of specifically regulating FSH release by the pituitary. However, inhibin and the related activins seem to play important paracrine roles in locally regulating testicular and ovarian function. They are also involved in paracrine regulation in the placenta, the pituitary, and other nonreproductive tissues. The local effects of these factors may be modified by the presence of binding proteins and the receptor or receptors expressed.The specific type of activin or inhibin produced and the net effect locally may vary as a function of the sexual development of the organism or the stage of the reproductive cycle. A more complete understanding of these complex relationships will develop with advances in our ability to measure the proteins, their binding proteins, and cell surface receptors.

The steroid receptor superfamily: mechanisms of diversity

The steroid receptor superfamily of ligand-dependent transcription factors is characterized by marked conservation of both structure and function between the various receptors. Despite their well-documented extensive similarities, these receptors respond to a diverse range of ligands, which results in an even more impressive diversity of function. A variety of strategies is used at each point in the pathway from ligand binding to gene expression to achieve this diversity. The nature of the ligand is important as are the tissue-specific patterns of receptor gene expression, the presence of binding proteins, and the effects of cell- or tissue-specific ligand-modifying enzymes. Once bound to the receptor, the nature of which may vary as a result of either differential splicing or gene duplication yielding multiple isoforms, the activated receptor may form hetero- or homodimers. A complex interplay then occurs between the receptor dimer, other nuclear proteins, the response element, and the promoter complex to regulate gene expression. These elements may vary as a function of the cell type, other stimuli, and the context and sequence of the response element (or elements) in a given gene. By these mechanisms diversity may even be achieved for a given ligand, receptor subtype, gene, or cell. The observations may help to explain certain phenomena in hormone biology that are difficult to reconcile with the previous, simple, univariant model of steroid hormone action.

Failure of acid–ethanol treatment to prevent interference by binding proteins in radioligand assays for the insulin-like growth factors

Acid-ethanol precipitation and gel filtration at acidic pH have been widely used to extract circulating binding proteins for insulin-like growth factor (IGF-I and IGF-II) from plasma or serum samples before radioligand assay for the respective IGFs. Gel filtration on Sephadex G-50 at neutral pH of neutralized acid-ethanol extracts of fetal and adult ovine plasma which had been incubated with 125I-labelled IGF-I or 125I-labelled IGF-II revealed that significant amounts of the IGF-binding protein activity survived the acid-ethanol extraction procedure. Radioimmunoassay for IGF-I in acid-ethanol extracts of plasma samples from fetal, neonatal and adult sheep yielded results which depended upon the method used for separation of the antibody-bound IGF-I tracer from the free IGF-I tracer. Acid gel filtration of ovine fetal and adult plasma was found to remove completely the IGF-binding protein activity. Radioimmunoassay for IGF-I in samples of fetal, neonatal and adult sheep plasma that had undergone acid gel chromatography yielded consistent results for both methods that were used to separate antibody-bound IGF-I tracer from the free tracer. Radioreceptor assays for IGF-II were similarly highly perturbed by the presence of binding protein in acid-ethanol extracts of ovine fetal and adult plasma. We conclude that acid-ethanol extraction can not be used reliably for the removal of IGF-binding proteins, and that only acid gel filtration is a completely safe and valid method.

15] Two-site enzyme immunoassay for nerve growth factor

Publisher Summary This chapter describes immunological determinations of nerve growth factor (NGF), although it should be noted that probably as many difficulties occur in the determination of its biological activity. The enzyme immunoassay presented in the chapter is sufficiently sensitive to analyze the physiologically relevant NGF levels. The use of both mono- and polyclonal anti-NGF antibodies together with the use of nonspecific antibody preparations as background control is essential to ensure the specificity of the NGF determination with the two-site enzyme immunoassay. Many false-positive NGF determinations have been obtained using competition assays. Such results could not be confirmed with both reliable two-site immunoassays and biological assay methods. The difficulties encountered in NGF determinations are because of the presence of NGF-binding proteins and the strong nonspecific adsorptivity of NGF. These difficulties become significant at the extremely low NGF levels in tissues in which NGF has a physiological role. Problems arising from the presence of binding proteins are not unique for NGF, but have also been reported for other rare proteins such as platelet-derived growth factor.

Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus

A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus. The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein. The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl. The isolated protein can bind to both circular and linear duplex DNA. Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients. The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template. The binding protein itself is free of detectable DNA polymerase or nuclease activity.

Properties of the Escherichia coli DNA-binding (unwinding) protein interaction with nucleolytic enzymes and DNA

The Escherichia coli DNA-binding protein reduces the activity of most single-stranded DNases from a variety of sources; however, the nucleases associated with E. coli DNA polymerase II and bacteriophage T7-induced DNA polymerase and E. coli exonuclease I remain active in the presence of binding protein and may even be stimulated. Those nucleases whose activities are stimulated by binding protein interact with the protein in the absence of DNA, to give a specific enzyme-binding protein complex, e.g. E. coli DNA polymerase II and E. coli exonuclease I both form a species, the sedimentation of which is compatible with a complex of one enzyme molecule plus one or two monomers of binding protein. Furthermore, the apparent affinity of nucleolytic enzymes for their substrate, single-strand DNA, is altered by complexing the DNA with binding protein. In particular, DNA polymerase II binds in a stable way only to DNA that is saturated with binding protein. This facilitation of binding to DNA led to the observation that in contrast to the mechanism of digestion of free DNA, E. coli DNA polymerase II digests DNA complexed with DNA-binding protein that is in a manner in keeping with a processive mechanism.

A GROWTH HORMONE BINDING PROTEIN IN NORMAL HUMAN SERUM.

IT has been demonstrated that human growth hormone migrates in agar gel electrophoresis at the rate of the α-globulins1. This occurs both in pure solution and in serum. The technique of radioimmunoelectrophoresis has now been used to demonstrate the presence of binding proteins for both insulin2 and thyroxine3. In this communication the technique is applied to human growth hormone.