Search

Publisher Summary This chapter presents an overview of the techniques that are available to study the interaction of protein phosphatase 1 (PP1) with other proteins. Most interactors of PP1 are inhibitors of the phosphorylase phosphatase activity of the catalytic subunit. A simple phosphatase assay can give an independent indication that the studied polypeptide is indeed an interactor of PP1. It discusses another technique known as “PP1 overlays,” based on the binding of PP1 to interactors that are associated with PVDF-membranes. PP1 overlays can be used to demonstrate a direct interaction between PP1 and a putative interactor in a complex mixture of proteins. The binding between PP1 and its interactors can also be explored by pull-down experiments when one of the two components is fused to gluthathione S-transferase (GST), a protein that binds with high affinity to glutathione-agarose. An occasional problem with GST pull-down experiments is the high background, because of the nonspecific binding of the putative PP1 interactor to GST. Another classical approach to demonstrate an interaction between two proteins is their coimmunoprecipitation from cell extracts. Some other tools are described in the chapter to study the interaction between PP1 and associated polypeptides.

Publisher Summary This chapter describes the purification from rabbit skeletal muscle of an enzyme complex of Mr 70,000, which accounts for most of the total phosphorylase phosphatase activity in crude extracts. The enzyme is a heterodimer consisting of two subunits: an Mr 38,000 catalytic subunit and an Mr 31,000 regulatory subunit that is identical to inhibitor-2. The enzyme as isolated is totally inactive but can be activated in two ways. First, full activation can be obtained by limited proteolysis with trypsin in the presence of Mn2+. Second, the enzyme can be activated by an ATP. Mg2+-dependent reaction first described by Merlevede and Riley that also requires a factor known as Fa that appears to be identical to glycogen synthase kinase-3. The purification procedure is essentially that used by Ballou et al. and involves DEAE chromatography, acetone precipitation, gel filtration, and finally affinity chromatography using polylysine as the bound ligand. Throughout the procedure, the phosphatase is monitored by its activity toward phosphorylase a as substrate. Several other similar procedures for the isolation of the Mr 70,000 ATP Mg2+-dependent phosphatase are also described in the chapter.

Publisher Summary Adenosine 5'-O-(γ-thio)-triphosphate (ATPγS) can replace ATP as an effective substrate of many kinases, particularly protein kinases. On the other hand, ATPγS is hydrolyzed very slowly by phosphatases and by most ATPases in comparison with ATP. Similarly, proteins thiophosphorylated by ATPγS are also resistant to protein phosphatases. Thus, thiophosphorylation of phosphorylase b by phosphorylase kinase leads to its conversion to the activated a form that is resistant to dethiophosphorylation and inactivation by phosphorylase phosphatase activity. The metabolic stability of thiophosphorylated proteins has also been demonstrated for myosin light chain in smooth muscle contraction, and for the receptor of epidermal growth factor in the epidermal carcinoma cell line A431. Therefore, thiophosphorylation of protein by ATPγS can serve as a powerful tool for investigating the regulatory mechanism of enzymes and metabolic processes that are controlled by a cycle of protein phosphorylation and dephosphorylation.

Publisher Summary This chapter describes properties of fat body glycogen phosphorylase in relation to flight directed carbohydrate mobilization in locusts. It presents a study that deals with some properties of phosphorylase from fat body of Locusta migratoria and its regulation by hormone(s) from the corpora cardiaca (CC). Locust fat body contains active phosphorylase a (AMP-independent) and inactive phosphorylase b (AMP-dependent). Results of various incubation experiments with crude extracts of fat body showed the presence of phosphorylase b kinase activity, being ATP and Mg 2+ dependent, and of phosphorylase phosphatase activity that is inhibited by NaF. Each of these enzymes may be involved in the regulation of phosphorylase activity and, thus, of the rate of glycogenolysis. Injection of locusts with CC-extract results in the activation of fat body phosphorylase. At least, a part of this effect may be attributed to adipokinetic hormone (AKH) produced by the CC as shown by the injection of synthetic AKH. As little as 0.02 pmol AKH—corresponding with 0.0001 pair CC equivalents—causes a significant increase of phosphorylase activity compared with water-injected controls.

Publisher Summary This chapter describes properties of protein phosphatase C. Phosphorylase phosphatase/ Protein Phosphatase C was discovered as an enzymatic activity in rabbit muscle extracts, which catalyzed the conversion of phosphorylase a to phosphorylase b. The nature of the interconversion was only later shown to involve phosphorylation and dephosphorylation of phosphorylase. Attempts were made to isolate the enzyme from rabbit liver. It was discovered that treatment of rabbit liver extracts with high concentrations of ethanol at room temperature led to the activation of phosphorylase phosphatase activity and its conversion to a discrete lower molecular weight form of Mr 35,000. This low molecular weight form was readily amenable to further purification and a procedure was developed which allowed the separation of the enzyme in a homogeneous form from rabbit liver. The enzyme was characterized as having a molecular weight of ca. 35,000 by gel filtration and by SDS disc gel electrophoresis. The procedure also allowed the purification of the enzyme from rabbit skeletal muscle and from bovine heart. This Mr 35,000 phosphatase appears to be distributed in both liver and muscle tissues, and it is also present in tissues from different mammalian species.