The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-I , has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22 835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Straphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e. g. 1 -49, 49 -92, 67 ~ 101, 108 -134, 142 -182 and 163 -197) are inactive. Digestion with clostripain generated a peptide comprising residues 25 -114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-I .In 1976, Huang and Glinsmdnn [l] identified two proteins in rabbit skeletal muscle, termed inhibitor-I (1-1) and inhibitor-2 (I-2), that were specific inhibitors of a protein phosphatase, subsequently termed protein phosphatase-1 (reviewed in [2, 31). 1-1 was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase, whereas the activity of 1-2 was unaffected by this protein kinasc [I].1-2 can interact with protein phosphatase-I in two distinct ways. Firstly, at extremely low concentrations (Kd z 0.1 nM), it combines with the catalytic (C) subunit of protein phosphatase-I to produce an inactive 1-2 . C complex [4, 51, termed protein phosphatase-lI [6]. This form has also been termed the Mg-ATP-dependent protein phosphatase [7] because its activation requires preincubation with Mg-ATP and a protein kinase, termed factor FA [8] or glycogen synthase kinase-3 [9, 101. Activation of protein phosphatase-lI is triggered by the phosphorylation of a specific threonyl residue on 1-2 [4, 5, 11 -131 and is the first example of a protein phosphatase that is activated by a protein kinase. Secondly, at higher concentrations ( K d E S nM), 1-2 inactivates protein phosphatase-I by a different mechanism that is not reversed by preincubation with Mg-ATP and glycogen synthase kinase-3, and presumably results from the binding of 1-2 at a separate site on the enzyme [S].1-2 is also a substrate for other protein kinases, such as casein kinase-I1 [I41 and the insulin receptor [15]. Phosphorylation by casein kinase-11, which occurs on seryl residues, does not activate protein phosphatase-lI directly, but enhances the rate of phosphorylation of 1-2 and activation of protein phosphatase-1, by glycogen synthase kinase-3 [I 41. Phosphorylation by the insulin receptor occurs on a tyrosyl residue(s), but the functional significance of this modification is unknown.1-1 and 1-2 have been purified...

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptible to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the--AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH--group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.