Cindy Putnam-Evans

Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin

Calmodulin from both animal and plant sources is known to bind a number of hydrophobic compounds with resultant inhibition of calmodulin function. Some of these compounds, including certain phenothiazine and naphthalene sulfonamide derivatives, have been previously shown to be useful in the chromatographic isolation of calmodulin, when covalently linked to a solid support. With the exception of fluphenazine linked to epoxide-activated Sepharose, these resins have the undesirable characteristics of requiring high salt concentrations in the elution buffer for efficient elution of calmodulin, thus decreasing the selectivity for this protein. The synthesis of nine Sepharose-ligand affinity resins is reported. Some of the ligands are newly synthesized naphthalene sulfonamide and phenothiazine derivatives. The synthetic ligands have been coupled to three types of Sepharose: epoxide-activated, CNBr-activated, and carbodiimide-activated. The properties of these resins are reported and their relative abilities to act selectively in the isolation of calmodulin are compared. 2-Trifluoromethyl-10-aminopropyl phenothiazine (TAPP), when linked to epoxide-activated Sepharose, was found to be the most useful for calmodulin isolation in terms of its combined stability, capacity, and ability to select for calmodulin. This resin was found to behave as a true affinity resin. A quantitative evaluation of its affinity behavior was consistent with the presence of two high-affinity Ca2+-dependent phenothiazine binding sites on calmodulin, in apparent agreement with previous reports which involved the use of different methods.

Calcium-Dependent Protein Phosphorylation in Suspension-Cultured Soybean Cells

The discovery of the calcium-binding protein calmodulin in plants (Anderson and Cormier, 1978; Anderson et al., 1980) provided the basis for suggesting that Ca2+ serves a second messenger role in plants, and that Ca2+-dependent metabolic regulation in plant cells may be mediated by such Ca2+-binding proteins (Anderson et al., 1980). Support for these hypotheses has come from the demonstration that enzymes such as pea NAD kinase (Anderson and Cormier, 1978; Anderson et al., 1980) and Ca22+-transport ATPases of zucchini (Dieter and Marme, 1980) and corn (Dieter and Marme, 1981) are activated by calcium and calmodulin. Recently, several investigators have observed calcium-dependent and possibly calmodulin-dependent phosphorylation of endogenous proteins in plant extracts (Hetherington and Trewavas, 1982; Salimath and Marme, 1983; Veluthambi and Poovaiah, 1984a; 1984b; Putnam-Evans and Cormier, 1984). Also, Ca22+-dependent protein kinases have been partially purified from wheat germ (Polya and Davies, 1982 Polya et al., 1983; Polya and Micucci, 1984) and soybean cells (Putnam-Evans and Cormier, 1984). The regulation of protein phosphorylation by calcium may be a mechanism of metabolic and physiological control in plants, as it is in animals.

[22] Directed mutagenesis in photosystem II: Analysis of the CP 47 protein

Publisher Summary This chapter describes the techniques used in laboratory to introduce directed mutations into the chlorophyll-protein CP 47 of the cyanobacterium Synechocystis 6803. This protein is an integral membrane component of the proximal chlorophyll a antenna of PS II, which also interacts with the oxygen-evolving site. The chapter investigates the roles of the conserved, charged residues located principally in the large extrinsic loop E of CP 47, which appears to be lumenally exposed. This domain is examined using a variety of biochemical techniques and there exists a strong body of evidence indicating that this portion of CP 47 interacts with components required for oxygen evolution. Site-directed mutations were introduced into this region of CP 47 using the method of Kunkel. Random mutations directed against the large extrinsic loop domain of CP 47 are introduced in the chapter using the mutator strain of E. coli XL-1 Red.