Self-association of phosphorylase kinase from rabbit skeletal muscle in the presence of natural osmolyte, trimethylamine N-oxide
The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8 and 8.2. PhK is a hexade-camer (αβγδ)4 with a molecular mass of 1,300 kDa. The oligomeric state of the native enzyme is dependent on the protein concentration and the concentrations of Ca2+ and Mg2+, which are essential for the enzymatic activity. In the absence of Ca2+ and Mg2+ the enzyme exists in the monomeric and dimeric forms (with s 20,w = 23 and 36.5 S); however, the addition of 0.1 mM Ca2+ and 10 mM Mg2+ results in the appearance of associates of higher order. TMAO (0.6–1.0 M) was found to favor greatly self-association of PhK. In the presence of TMAO, apart from the association products, consisting of a rather low number (n) of PhK molecules (n = 2, 3, 4,...), two distinct rapidly moving boundaries with S 20,w = 189 and 385 S are registered on the sedimentation profiles. These boundaries correspond to 24-mers and 70-mers (the molecular masses of associates were estimated using the Mark-Houwink—Kuhn—Sakurada equation, assuming a spherical form. The kinetics of TMAO-induced association of PhK was monitored by following the increase in the turbidity of the enzyme solution at varying concentrations of the protein and TMAO. The initial rate of the association is proportional to the enzyme concentration squared, suggesting that the initial step of PhK association is the stage of dimerization.
Key wordsAnalytical ultracentrifugarion Self-association Phosphorylase kinase Trimethylamine N-oxide Crowding
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