Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].
Fibrinogen adsorption from plasma exhibits an unusual displacement phenomenon (the Vroman effect) in that decreases in adsorption occur after longer contact time or as the plasma concentration increases. Fibrinogen adsorption and the displacement effect were found to depend markedly on the nature of the surface, the time and temperature of adsorption, and to a lesser extent on certain contact activation factors. Displacement still occurred from plasmas lacking kininogens, factor IX, and prekallikrein, and from plasma that had been treated with BaSO4 to remove factors II, VII, IX, and X. Furthermore, displacement was also observed from fibrinogen solutions to which either albumin or hemoglobin had been added. In addition, competitive adsorption of binary protein mixtures was also shown to depend strongly on surface type. It therefore appears that fibrinogen adsorption from plasma is subject to similar if not identical competitive processes that occur in simpler protein mixtures. The final adsorption then reflects the influence of all the proteins in plasma, each competing for the limited number of adsorption sites according to the fundamental physical properties of surface activity and mass concentration.
A series of hydrogels with large pores was synthesized by the precipitation polymerization of 2-hydroxyethyl methacrylate (HEMA) with crosslinking agent in aqueous solution. Such gels are potentially useful for the controlled release of large-molecular-weight species such as proteins. In this study, the release behavior of lysozyme and alpha-amylase from hydrogels formed from HEMA or HEMA with a comonomer was studied. It was found that the polymer composition affected the total amount of lysozyme released and its activity. Effects were smaller with alpha-amylase. Charged gels, containing a phosphate moiety, released larger amounts of lysozyme at a reduced rate as a result of charge-charge interactions.
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