Search PubMed⌕ Search

Biomedical subjects

D F Senear

Publications and source records attributed to D F Senear.

27 records · Page 2Linked to original sources
PubMed1986

Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding.

Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Bacteriophage lambda↗
PubMed1986

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins.

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.

Amino Acid Sequence↗
PubMed1986

"Footprint" titrations yield valid thermodynamic isotherms.

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.

Binding Sites↗
PubMed1985

Multiple ion-dependent and substrate-dependent Na+/K+-ATPase conformational states. Transient and steady-state kinetic studies.

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway.

Adenosine Triphosphate↗
PubMed1983

Current status of the molecular structure and function of the plasma sex steroid-binding protein (SBP).

Purification and characterization of the sex steroid-binding protein (SBP) from human, macaque, baboon, and rabbit sera indicate that the protein is composed of two polypeptide chains which associate noncovalently to yield a native structure having molecular weight distributions of about 88,000 for primate SBPs, and 80,000 for rabbit SBP. The subunit molecular weight distributions are 44,000 for human SBP, 47,000 for macaque and baboon SBP's, and 40,000 for rabbit SBP. Isoelectric focusing show extensive microheterogeneity for all four SBPs. The patterns appear to be unique for each species and reveal the presence of at least twelve bands of different colour intensity reflecting a specific spectrum of active SBP molecules. The existence of the large number of dimeric forms of SBP arises through the combination of many variants of the same two subunits containing different amounts and types of carbohydrate sidechains. Physiological studies on the intravenous infusion of pure rhesus SBP, human SBP, and purified monospecific SBP-antibodies into the rhesus reveal an inverse relationship between SBP and the metabolic clearance rate of testosterone. The effect is complex and depends on the concentration of SBP, albumin, and testosterone which in turn influences the distribution of testosterone between albumin and SBP.

Animals↗
PubMed1981

Thermodynamics of concanavalin A dimer-tetramer self-association: sedimentation equilibrium studies.

The effects of temperature and pH on the reversible dimer-tetramer association of concanavalin A were studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified preparations of concanavalin A were used. Equilibrium constants were analyzed as a Wyman linked function by using truncated van't Hoff temperature dependence. In the concentration range 0.1-3.0 mg/mL, from 5 to 35 degrees C and between pH 5.5 and pH 7.5 at 0.5 M ionic strength, only dimer and tetramer species were present in both preparations. For purified concanavalin A, association constants ranged from 1.5 X 10(3) to 8.0 X 10(7) M-1. Constants for our commercial preparation were approximately 10-fold lower due to the decrease competency of some subunits to self-associate. From the fit of the Wyman model to the experimental data, delta G degree, delta H degree, delta S degree, and delta Cp degree were calculated for the association and association-linked ionization reactions. From the values of the ionization thermodynamic parameters, the association is governed by the ionization of a histidine side chain on each subunit, either histidine-51 or histidine-121. The association is characterized by large entropy (66.3 cal.mol-1.deg-1 at 25 degrees C) and heat capacity (-821 cal.mol-1.deg-1) changes in accordance with the large hydrophobic association surface observed in crystallographic studies [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547]. In addition, there is a large enthalpy change (10.4 kcal.mol-1 at 25 degrees C). We propose a model for the interaction based on a more detailed thermodynamic description than was obtained in an earlier, incomplete study [Huet, M., & Clavarie, J. M. (1978) Biochemistry 17, 236-241].

Concanavalin A↗
PubMed1981

Effects of saccharide and salt binding on dimer-tetramer equilibrium of concanavalin A.

The effect of the binding of saccharide ligands on the reversible dimer-tetramer equilibrium on concanavalin A was studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified fragment-free concanavalin A preparations were used. In the case of the fragment-free preparation, there was no effect of the binding of alpha-methyl mannoside or alpha-methyl glucoside at 35 degrees C and at a variety of conditions of pH and ionic strength. This implies no difference in ligand binding activity between dimeric and tetrameric Con A, in contrast to an earlier report [McKenzie, G. H., & Sawyer, W. H. (1973) J. Biol. Chem. 248, 549-556]. There was a profound effect in the case of the commercial preparation. Dimers that contain hydrolyzed subunits appear to be incompetent to self-associate in the presence of alpha-methyl mannoside or alpha-methyl glycoside, while alpha-methyl galactoside, which does not bind to Con A, had no effect. The effects of very high concentrations of CaCl2 (to 2.5 m) and NaCl (60 6.2 m) were also studied. The data were analyzed by an integrated form of the Tanford extension [Tanford, C. (1969) J. Mol. Biol. 39, 539-544] of the Wyman linked function theory, which includes preferential interactions with salt and water. The integrated form allows preferential interactions to be described as the sum of salt binding and water binding. The data were well described by salt binding alone; it was unnecessary to invoke any water binding effect. The CaCl2 data did indicate that one calcium per subunit of the dimer binds to a site that is buried in the tetramer. This suggests a site on the dimmer-dimer interface which is consistent with Reeke's identification of the protomers composing the solution dimer [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547].

Calcium↗