Purification and some properties of rabbit skeletal muscle fructose 6-phosphate kinase inhibitor.
The loss of activity of rabbit skeletal muscle FPK on storage and its restoration by ATP, AMP and cyclic AMP has prompted us to look for an inhibitory unit of the enzyme. We have purified this inhibitory factor from the crude muscle extract and isolated from crystalline FPK; both proteins have the same Mw of about 68,000 (SDS). Carboxymethylation revealed species of lower molecular weight. It is suggested that two different kinds of FPK exist, one composed only of "active" subunits and another composed only of "inactive" (inhibitor) subunits. States of intermediate activity exist, created by dissociation, reassociation and exchange of subunits, because the inhibitor and FPK share several subunits. A model is proposed where one or several inhibitors of molecular mass 68,000 replace the corresponding number of active subunits of 93,000 daltons, the structure of the native molecule remaining tetrameric. It is shown that cyclic AMP exerts its activation function on FPK only in the presence of the inhibitory protein, probably by displacing the exchange of the subunit in favor of the active tetrameric species of 360,000. Ammonium chloride plays probably an opposite role in this exchange. The inhibitor coverts the Michaelian behavior with respect to F-6-P into a cooperative response (sigmoidal shape of the curve) characterized by a Hill coefficient of 2. The Michaelian response with respect to ATP is preserved, the corresponding constant being only slightly affected. In the presence of subsaturating concentration of inhibitor, mixed species are detected. As a first approximation one can propose that a reversible equilibrium exists between free and complex FPK subunits. The dissociation constant of this equilibrium being equal to 4 X 10(-8) M in moles of FPK protomers.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000502:physiology*; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis; D013997:Time Factors
D046911:Macromolecular Substances; D009124:Muscle Proteins; D013438:Sulfhydryl Compounds; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D010732:Phosphofructokinase-1