Electron microscopy of native and dimethylsuberimidate crosslinked rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP:D-fructose-6-phosphate I-phosphotransferase) has been carried out using negative staining with sodium phosphotungstate. The results obtained suggest the protomer of molecular weight 80,000 can be approximated as a prolate ellipsoid with axes of 67 A and 25 A. The dimer, which is the fundamental unit for polymerization, is formed by association along the 25 A axis and has approximate dimensions of 67 A X 55 X X 25 A. The tetramer appears to be formed by an end-to-end aggregation of dimers, and the octamer is a sheet-like structure made up of a side-to-side aggregation of tetramers. Higher crosslinked aggregates and long crosslinked filaments also are seen. The filaments have a constant width of about 250 A, are about 0.5 gm in length, and appear to involve tetramers as a fundamental structural unit. The functional significance of the filament structure is not known.Rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP:Dfructose-6-phosphate 1-phosphotransferase) plays an important role in the regulation of glycolysis. The steady-state kinetic properties of the enzyme suggest it is allosteric (1) and that the catalytic activity depends on the aggregation state of the protein (2, 3). The enzyme is made up of apparently identical subunits of approximately 80,000 molecular weight (4-6). Although the Stokes' radii and molecular weights of specific aggregates have been determined (2-5) and chemical crosslinking has been used to study the modes of aggregation (7), direct measurements of the size and shape of the fundamental subunit and its aggregates have not been reported. In the work reported here, electron microscopy has been used to obtain information about the sizes and shapes of the phosphofructokinase subunit of molecular weight 80,000 and its aggregates, both with native and chemically crosslinked enzyme.
EXPERIMENTAL SECTIONMaterials. The enzyme was prepared by the method of Ling et al. (8), as described (2). The final ammonium sulfate precipitate was dissolved in 0.1 M potassium phosphate (pH 8.0), 1 mM EDTA and dialyzed overnight against the same buffer to remove ammonium sulfate. The concentration of the enzyme stock solution was 10-14 mg/ml, and the specific activity of the enzyme at 230, pH 8.0 (33 mM Tris-HCl, 2 mM fructose 6-phosphate, 2 mM ATP, 5 mM MgCl2) was 120 units/mg. (A unit of enzyme activity is defined as the production of 1 ,umol of product per min.) The protein concentration was determined from the absorbance at 280 nm assuming an extinction coefficient of 1.02 ml/(mg-cm) (9), and a spectrophotometric assay using coupling enzymes was used (2, 8). The enzyme was crosslinked with dimethylsuberimidate, and specific aggregates were purified as described (7).The dimethylsuberimidate was synthesized from the suberonitrile (10), and all other chemicals were standard commercial materials.Electron Microscopy. The native enzyme was examined under conditions where it was primarily...