Abstract
A protein has been purified from rat liver (about 5 mg from 100 g) which inactivates rat liver phosphofructokinase-1. According to dodecyl sulfate gel electrophoresis the protein consists of a single peptide chain with a Mr of 19,000. The inactivation of phosphofructokinase-1 by this protein results from a dissociation of phosphofructokinase-1 into its inactive protomers (Mr = 82,000). The inactivation is dependent on zinc ions in micromolar concentration (about 1-2 microM), but is inhibited by higher concentrations (greater than 50 microM). Fructose 1,6-bisphosphate as well as fructose 2,6-bisphosphate inhibit the inactivation reaction. In addition, both compounds as well as ATP can reverse the dissociation of phosphofructokinase-1. The phosphofructokinase-1 inactivating protein has no phosphatase activity with [32P]phosphofructokinase or low molecular weight phospho-compounds and does not possess any detectable proteolytic activity. It has the same affinity for the phospho- and the dephosphoform of phosphofructokinase-1, but preincubation of phosphofructokinase-1 with this inactivating protein reduces the maximum amount of phosphate incorporated into phosphofructokinase-1 and accelerates the velocity of the dephosphorylation reaction. A direct Zn2+-dependent binding of phosphofructokinase-1 to the inactivating protein has been demonstrated in experiments with matrix-bound phosphofructokinase-1 inactivating protein.
Highlights
Brand and Hans-Dieter Soling From the Abteilunc-l.fiir Klinische Biochemie
A protein has been purified from rat liver
In 1976, we described a protein from-rat liver cytosol which inactivated
Summary
"One unit is defined as the activity which leads to the loss of 1 unit of phosphofructokinase-1 activity during 1min. Purified phosphofructokinase-1 inactivating protein (1.05 mg, 40 units/mg) in 2 ml of 20 M triethanolamine HCl, pH 7,4, was combined with 0.5 ml of settled Affi-Gel 102, and the pHwas adjusted to 5.0. The degree of binding of the protein to thematrix was measured by analyzing the supernatantfor unbound inactivating protein by SDS-PAGE. The molecular weight of the phosphofructokinase-1 inactivating protein was determined on 10-20% polyacrylamide gradient gels; proteolytic degradation of phosphofructokinase-1 was examined with 1%gels. Protein concentrationswere measured by the Coomassie Blue binding assay [22] using bovine serum albumin as a standard. Purified phosphofructokinase-1 inactivating protein was determined after acid hydrolysis by the ninhydrin reaction, as this protein did not react with Coomassie Blue G or Folin-Ciocalteu's reagent
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