Crystallization and Properties of Rabbit Skeletal Muscle Phosphofructokinase

From the ¹ From the Departments of Biochemistry and Biological Structure, University of Washington School of Medicine, Seattle, Washington 98105

Rabbit skeletal muscle phosphofructokinase was isolated and crystallized in the presence of adenosine triphosphate. The enzyme was homogeneous on electrophoresis. In the ultracentrifuge its behavior depended upon the conditions employed. For example, in glycerophosphate buffer at concentrations of enzyme above 0.1% a single asymmetric peak, s_20,w = 37 S, with a trailing edge was present. With decreasing protein concentrations the sedimentation coefficient dropped to an s_20,w of 13.8 S determined by density gradient centrifugation. In the presence of ATP lower sedimentation coefficients were observed, i.e. s_20,w = 29 and 8.4 S, respectively, under the above conditions. In p-hydroxymercuribenzoate, phosphofructokinase sedimented as a single component with an s_20,w = 13.1 S. The enzyme was stable when stored in the presence of ATP but aggregated in the absence of this nucleotide. It was determined that phosphofructokinase binds 1 mole of ATP, ADP, AMP, or cyclic 3',5'-AMP per 75,000 g of protein. The amino acid composition and the absorption spectrum of crystalline phosphofructokinase were determined. The crystalline enzyme showed the typical kinetic behavior previously reported for less pure preparations of enzyme in that it was inhibited by ATP and the inhibition was prevented by AMP or cyclic 3',5'-AMP. Phosphofructokinase examined in the electron microscope by negative staining revealed the presence of double or paired particles having dimensions of 130 x 100 x 25 A for the pair.

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