Febs Letters Volume 127, Number 1-2./Volume 128, Number 1-2. [antikvár]

Volume 127, number 1FEBS LETTERSMay 1981CONVERSION OF ACTIVE PROTEIN PHOSPHATASE TO THE ATP-Mg-DEPENDENT ENZYME FORM BY INHIBITOR-2Jackie R. VANDENHEEDE, Jozef GORIS, Shiaw-Der YANG, Theo CAMPS and WUfried MERLEVEDE Afdeling Biochemie, Departement Humane Biologie, Faculteit der Geneeskunde, Katholieke Universiteit te Leuven, BelgiumReceived 10 March 19811. IntroductionBrandt et al. [1,2] made the original observation that liver and muscle extracts contain heat-stable, trypsin-labUe proteins which inhibit phosphorylase phosphatase. The regulatory role of these heat-stable proteins in the control of phosphatase activity was accentuated when two heat-stable inhibitors, termed inhibitor-1 and -2 were discovered and the inliibitory capacity of inhibitor-1 demonstrated to depend upon its phosphorylation by the cyclic AMP-dependent protein kinase [3,4]. Heat-stable phosphatase inhibitors have since been identified in a variety of animals and tissues [510] and purified to homogeneity from rabbit muscle (inhibitor-1, -2) [8,10] and rabbit liver [7]. The implication of inhibitor-1 in the hormonal regulation of glycogen metabolism has been clearly demonstrated [11-13].We have reported the purification and characterisation of an ATP-Mg-dependent protein phosphatase system from rabbit muscle [14-19]: an inactive multifunctional protein phosphatase was shown to be activated by another protein factor (F^) in the presence of ATP-Mg ions (without phosphorylation). This report provides evidence that purified inhibitor-2 preparations [10] have the capacity to reverse the F^ and ATP-Mg-mediated activation.2. Materials and methodsMost materials and methods have been describedAddress correspondence to: Professor Dr W. Merlevede, Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgiumin [ 14,17]. The ATP-Mg-dependent phosphatase activity was commonly measured after a 10 min preincubation at 30°C with the activating protein F^^, 0.1 mM ATP and 0.5 mM Mg-acetate. One unit of phosphatase activity releases 1 nM [^^P]phosphate/min at 30°C from ^^P-labelled phosphorylase a (2 mg/ml).liihibitor-2 was purified to homogeneity from rabbit muscle according to [10]: specific activity was 80 000 U/mg. One unit of inhibitor-2 measured according to [ 10], decreased the activity of the ATP-Mg-dependent phosphatase [14] by 50%. The ATP-Mg-dependent phosphatase and protein phosphatase-1 have the same sensitivity towards 'Jte heat-stable protein inliibitors [19].Purified rabbit muscle inhibitor-1 (phosphorylated) was a generous gift of Dr P. Cohen, University of Dundee.Rabbit muscle protein phosphatase, F^ (10 000 U/mg) and the activating protein F^ (5000 U/mg) were purified according to [1] and [2], respectively.TPCK-treated trypsin was purchased from Worthington Biochemical Co. (England) and soybean trypsin inhibitor from Sigma Chemical Co. (USA).3. Results and discussion3.1. Preparation of trypsin-treated activated F^,-enzymeAbout 20 000 U Fc (2 mg protein) were fully activated by F^ and ATP-Mg and subsequently incubated at 30°C for 5 min with 0.2 mg TPCK-treated trypsin/ml. The proteolysis was stopped by the addition of 1.0 mg soybean trypsin inhibitor/ml and the 'irreversibly activated F(;-enzyme' applied to a (1 X 1 cm) polylysine-Sepharose 4B column equilibrated in a 20 mM Tris, 0.5 mM dithiothreitol pH 7Elsevier/North-Holland Biomedical Press1