Subunit interaction of rabbit muscle phosphofructokinase: effects of purification procedures.
Structural, physical, and kinetic properties of rabbit muscle phosphofructokinase (PFK) purified by three different procedures were monitored in order to determine the effect of various purification procedures on the dynamics of subunit interaction. PFK was purified by three commonly used procedures: (1) differential heat precipitation [Kemp, R. G. (1972) Methods Enzymol. 42, 71-77], (2) differential heat and alcohol precipitation [Ling, R. H., Marcus, F., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1893-1899], and (3) differential salt fractionation [Hesterberg, L. K., & Lee, J. C. (1980) Biochemistry 19, 2030-2039]. The physical, kinetic, and structural properties of these three preparations show that these proteins are not identical. Sedimentation velocity studies show that PFK purified by method 3 self-associates rapidly and that the system is thermodynamically homogeneous. The presence of an inactive or noninteracting component is not observed within an 8-h time limit. In contrast, PFK purified by method 1 or 2 is heterogeneous. In these preparations, a slowly sedimenting, noninteracting, inactive form of PFK is present. The remaining active protein is not stable but continuously converts to an inactive form. Active PFK can be fractionated from this inactive form by sedimentation. This active fraction behaves as a thermodynamically homogeneous system, and the subunits undergo rapid association-dissociation in a manner similar to PFK purified by method 3. Kinetic studies on these three preparations show that the inclusion of a heat and/or alcohol step in the purification procedure yields an enzyme that is less stable, has a lower specific activity, requires DTT for full activation, and is more susceptible to inhibition by ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Luther
M A
MA
Hesterberg
L K
LK
Lee
J C
JC
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010732:Phosphofructokinase-1 / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010732:Phosphofructokinase-1