(1/1199) Hydrophobic interaction of human, mouse, and rabbit interferons with immobilized hydrocarbons.

Interferons of human, mouse, and rabbit origin bind to straight chain hydrocarbons immobilized on agarose. The hydrophobic nature of binding is established by the following observations: (a) a positive correlation between the length of hydrocarbon ligand and the strength of interaction; (b) a stronger interaction with hydrocarbon ligands terminated with apolar rather than polar head groups; (c) a lack of dependence of binding on ionic strength and pH of the solvent; (d) a reversal of binding by ethylene glycol, a hydrophobic solute; (e) an increasing eluting efficacy of tetraalkylammonium ions with the length of their alkyl substituents. The hydrophobic interactions of human interferon underlie the efficiency of two-step chromatographic procedures. For example, human embryo kidney interferon can be purified about 3,600-fold by sequential chromatography on (a) concanavalin A-agarose, (b) octyl-agarose. Another two-step procedure: (a) concanavalin A-agarose, (b) L-tryptophan-agarose, gives about 10,000-fold purification. The overall recovery of interferon in both cases in close to 90%.  (+info)

(2/1199) Analysis of the stimulation-inhibition paradox exhibited by lymphocytes exposed to concanavalin A.

High doses of Concanavalin A (Con A), which normally inhibit T-lymphocyte stimulation as measured by increases in DNA synthesis, cause these lymphocytes to become committed to mitogenesis while also generating a dominant but reversible negative growth signal. The observed response to the stimulatory signal as measured by the rate of commitment to enter the S phase (i.e., the rate at which the stimulation becomes lectin independent) increases with lectin concentration even in the inhibitory range. The generation of this positive signal is prevented by treating the cells with colchicine. Cells that have become committed but are also simultaneously blocked from entering the S phase by the high doses of Con A can begin synthesizing DNA if the lectin is released by adding a competitive inhibitor of binding. Experiments done in agarose cultures in which lymphocytes are kept from contact with each other suggest that the reversible inhibitory signal is mediated by structures in the individual cells rather than as a result of agglutination. Continuously dividing cells of the lymphoid line P388 are also individually and reversibly inhibited by Con A. These findings are considered in terms of the relation of the inhibitory signal to the microtubular components of cell surface modulating assemblies made up of submembranous arrays of microtubules, microfilaments, and associated proteins.  (+info)

(3/1199) Adhesion energy of receptor-mediated interaction measured by elastic deformation.

We investigated the role of receptor binding affinity in surface adhesion. A sensitive technique was developed to measure the surface energy of receptor-mediated adhesion. The experimental system involved a functionalized elastic agarose bead resting on a functionalized glass coverslip. Attractive intersurface forces pulled the two surfaces together, deforming the bead to produce an enlarged contact area. The Johnson-Kendall-Roberts (JKR) model was used to relate the surface energy of the interaction to the elasticity of the bead and the area of contact. The surface energies for different combinations of modified surfaces in solution were obtained from reflection interference contrast microscopy (RICM) measurements of the contact area formed by the bead and the coverslip. Studies with surfaces functionalized with ligand-receptor pairs showed that the relationship between surface energy and the association constant of the ligand binding has two regimes. At low binding affinity, surface energy increased linearly with the association constant, while surface energy increased logarithmically with the association constant in the high affinity regime.  (+info)

(4/1199) A new method of quantitative affinity chromatography and its application to the study of myosin.

A new method of quantifying the interactions between two or three components of an interacting system, one of which is insoluble, is described. The method differs from those previously applied to affinity chromatography systems in that it does not require that elution volumes be measured, but is instead dependent on measurements of the quantity of affinity-bound material. Theoretical expressions are derived for systems in which the acceptor is immobilized. Examples presented to illustrate the validity of the theory are of the latter type and are from studies on the myosin-adenosine nucleotide-PPi system. With Sepharose-myosin columns (myosin covalently coupled to CNBr-activated Sepharose) a dissociation constant of 1.8 muM for ATP4- was found. Data were also obtained under conditions that closely approximate to those found in vivo, i.e. on columns packed with a slurry of Sephadex G-50 and precipitated myosin filaments formed at low ionic strength. The binding of MgATP2-, MgADP-, ATP4- and MgPPi2- to "filamentous" myosin in both two- (myosin and nucleotide) and three- (myosin, nucleotide and PPi) component systems at different temperatures was studied and the dissociation constants obtained agreed well with previously published values. Except for the binding of ATP4- to filamentous myosin at 4 degrees when 85% of the protein was interacting with the nucleotide, much lower values for the number of available sites occupied by the nucleotides were as a routine found in this system. Although this apparent discrepancy is difficult to explain, it is not an anomaly of the theoretical approach and may reflect the present state of understanding of the myosin system.  (+info)

(5/1199) Structural heterogeneity of the binding sites of HSA for phenyl-groups and medium-chain fatty acids. Demonstration of equilibrium between different binding conformations.

A new facet of the very heterogeneous albumin molecule is described. Chromatography at pH 6-9 of human serum albumin (HSA) on a phenyl-sepharose column separates it into two nonconvertible conformations that are, in turn, in equilibrium with its binding and nonbinding forms. The hydrophobic interaction of HSA with phenyl-sepharose depends on ionic strength, pH, and time of contact with the immobilized ligand. Binding as a function of pH shows a minimum at pH 6.5, and the binding profile at pH 7-9 fits the titration of a weak monoprotic acid with a pKa of 7.3. There was no observable difference in the CD spectra or the masses of the two forms. The equilibrium between the albumin forms was examined under defined conditions and cannot be explained by a simple two-state model. Thus rechromatography of the nonbinding fraction derived from a sample in which 50% of the protein was originally retained resulted only in 10-20% bound protein. Correspondingly only 70-80% of the binding form was retained. A model explaining the observations can be derived if two species, I and II, exist in the solution, both being in an equilibrium with a binding and a nonbinding form, but in which I is not in equilibrium with II. The rate of conversion between the binding and nonbinding conformations was determined to be faster than 15 s at room temperature.  (+info)

(6/1199) Resolution and purification of histones on homologous series of hydrocarbon-coated agaroses.

Hydrophobic chromatography on alkyl-agarose columns has been applied to the fractionation of histones. This paper describes: (a) a two-column method for the resolution of whole histone from calf thymus into its five main components (H1, H2a, H2b, H3 and H4), (b) a rapid one-step procedure for the isolation of the H3 fraction from whole histone, (c) an alternative one-step procedure for the resolution of H3 and H2a (which co-elute during gel exclusion chromatography on Biogel P-60). These experiments are also used for gaining further insight into the mechanism of action of hydrocarbon-coated agaroses.  (+info)

(7/1199) Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium.

Dictyostelium expresses at least two proteins of the cyclin-dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81-85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.  (+info)

(8/1199) Identification of a nucleic acid binding domain in eukaryotic initiation factor eIFiso4G from wheat.

Higher plants have two complexes that bind the m7G-cap structure of mRNA and mediate interactions between mRNA and ribosomal subunits, designated eIF4F and eIFiso4F. Both complexes contain a small subunit that binds the 5'-cap structure of mRNA, and a large subunit, eIF4G or eIFiso4G, that binds other translation factors and RNA. Sequence-specific proteases were used to cleave native cap-binding complexes into structural domains, which were purified by affinity chromatography. We show here that eIFiso4G contains a central protease-resistant domain that binds specifically to nucleic acids. This domain spans Gln170 to Glu443 and includes four of the six homology blocks shared by eIFiso4G and eIF4G. A slightly shorter overlapping sequence, from Gly202 to Lys445, had no nucleic acid binding activity, indicating that the N-terminal end of the nucleic acid binding site lies within Gln170 to Arg201. The binding of the central domain and native eIFiso4F to RNA homopolymers and double- and single-stranded DNAs was studied. Both molecules had highest affinity for poly(G) and recognized single- and double-stranded sequences.  (+info)