Subject protein affinity

As another example, imagine that an S-100 fraction has been prepared, ammonium sulfate has been added, and the fraction that precipitates between 30 and 50% has been obtained. This sample is subjected to affinity and ion-exchange chromatography. Have these procedures been successful in removing extraneous proteins While it is possible to answer this question by a determination of total protein content, it may be more informative to analyze each of the samples for its constituent proteins. 

Proteins in the cell-free culture supernatant v re concentrated by ultrafiltration using a M-10 filter (Amicon). The retentate was subjected to affinity chromatography using regenerated chitin (Roberts and Cabib, 1982). Fractions v re tested for protein according to Bradford (1976) and by 280 nm absorbance. 

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

The cytoskeleton also contains different accessory proteins, which, in accordance with their affinities and functions, are designated as microtubule-associated proteins (MAPs), actin-binding proteins (ABPs), intermediate-filament-associated proteins (IFAPs), and myosin-binding proteins. This chapter is focused on those parts of the cytoskeleton that are composed of microfilaments and microtubules and their associated proteins. The subject of intermediate filaments is dealt with in detail in Volume 2. 

More than 50 proteins have been discovered in the cytosol of nonmuscle cells that bind to actin and affect the assembly and disassembly of actin filaments or the cross-linking of actin filaments with each other, with other filamentous components of the cytoskeleton, or with the plasma membrane. Collectively, these are known as actin-binding proteins (ABPs). Their mechanisms of actions are complex and are subject to regulation by specific binding affinities to actin and other molecules, cooperation or competition with other ABPs, local changes in the concentrations of ions in the cytosol, and physical forces (Way and Weeds, 1990). Classifications of ABPs have been proposed that are based on their site of binding to actin and on their molecular structure and function (Pollard and Cooper, 1986 Herrmann, 1989 Pollard et al., 1994). These include the following ... 

Protein phosphorylation-dephosphorylation is a highly versatile and selective process. Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a protein s surface, only one or a small subset are targeted. While the most common enzyme function affected is the protein s catalytic efficiency, phosphorylation can also alter the affinity for substrates, location within the cell, or responsiveness to regulation by allosteric ligands. Phosphorylation can increase an enzyme s catalytic efficiency, converting it to its active form in one protein, while phosphorylation of another converts it into an intrinsically inefficient, or inactive, form (Table 9—1). 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

MS can perform large-scale analyses of protein interactions. Interacting partners of proteins in complexes can be purified by several strategies including affinity chromatography and immunoprecipi-tation with antibodies specific to the bait protein. The purified components can then be subjected to LC-MS/MS and proteins within the complex identified. Using a variety of approaches including... 

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