Key points are not available for this paper at this time.
Inhibitor-2, a protein inhibitor of the multifunctional enzyme termed protein phosphatase-1 Cohen, P. (1978) Curr. Top. Cell. Regul. 14, 117-196, was purified by heat treatment at 90°C, precipitation with trichloroacetic acid, chromatography on DEAE-cellulose at pH 8.5, gel filtration on Sephadex G 150, and finally two successive chromatographic steps on DEAE-cellulose at pH 5.0. The protein was purified 13000-fold, and nearly 1.0 mg per 1000 g of muscle was obtained in eight days corresponding to an overall yield of 25%. The purified protein was homogeneous by the criterion of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Similar concentrations of inhibitor-2 were found in different skeletal muscle fibres and the molar concentration in vivo is 0.35 μM. The sedimentation coefficient, S20,w, determined by sucrose density gradient centrifugation was 1.75 S and the Stokes radius determined by gel filtration was 3.5 nm. These values, combined with the partial specific volume of 0.714, determined from the amino acid composition, yielded a molecular weight of 25500. The molecular weight determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was 30500. The sedimentation and gel filtration behaviour of the protein, its stability to a variety of denaturing conditions (heat, acid, urea and ethanol) and its very low content of hydrophobic amino acids, suggests that inhibitor-2 is not a globular protein. Inhibitor-2 did not contain significant amounts of covalently bound phosphate, and this was consistent with the finding that its activity was unaffected by pre-incubation with either adenosine-3′.5′-monophosphate-dependent protein kinase and ATP-Mg, or with protein phosphatase-1. 50% inhibition of protein phosphatase-1 was observed at 7.0 nM inhibitor-2, and the phos-phorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthase phosphatase activities of the enzyme were inhibited in a very similar manner. The properties of inhibitor-2 are compared with those of inhibitor-1 in the discussion.
Foulkes et al. (Sat,) studied this question.