Rabbit kidney cortex phosphorylase phosphatases: evidence for complexes between high molecular weight forms and heat-stable inhibitor proteins.

Abstract

Two forms of high molecular weight phosphorylase phosphatase have been partially resolved by gel filtration chromatography of rabbit kidney cortex extracts. Two heat-stable inhibitor proteins co-eluted with the phosphatase peaks. Phosphorylase phosphatase and heat-stable inhibitor activity also co-migrated on gel electrophoresis of cortex extracts. When extracts were heated to 95 degrees for 5 minutes prior to gel filtration or electrophoresis, phosphorylase phosphatase inhibitor activity eluted at a lower molecular weight and a higher mobility, respectively. Storing cortex extracts at -20 degrees for 6 months resulted in partial conversion of both phosphatase and inhibitor activities to lower molecular weight forms which co-eluted on gel filtration. The two inhibitor peaks from gel filtration chromatography were heat-treated and characterized. Both inhibitor peaks had molecular weight of 25,000 to 35,000. The inhibitory activity of one of the peaks was increased about 3.5-fold by incubation with cyclic AMP-dependent protein kinase and ATP, and required higher concentrations of TCA to be precipitated. Hence, one of the inhibitor peaks resembled rabbit muscle inhibitor -1, while the other peak may represent an inhibitor similar to rabbit muscle inhibitor -2. These results represent the first indication that low molecular weight heat-stable inhibitor proteins may be bound to high molecular weight phosphorylase phosphatases in the cell.