Identification and partial characterization of bovine heart cytosolic phosphorylase phosphatases

The properties of phosphatases in bovine heart cytosol were studied. Two isozymic forms of protein phosphatase H (H-1 and H-2) were resolved by chromatography on DEAE-Sephacel. The two isoenzymes had identical physical properties (Mr 260,000, 7.9 S). Treatment with 80% ethanol activated both isozymes and converted H-1 to a Mr 35,500 form and H-2 to Mr 67,000 and Mr 35,500 forms. Both H-1 and H-2 and their lower Mr activated forms had essentially identical Km values for phosphorylase a. The heart cytosol also contained a latent phosphatase (Fc) which could be activated by preincubation with either ATP X Mg and an activating factor (FA), or by Mn/trypsin treatment. The latter procedure converted the latent Fc (Mr 200,000) to a Mn2+-independent Mr 34,500 form. Both activated forms of Fc had similar Km values which were fourfold lower than the affinity of the protein phosphatase H forms for the phosphorylase a substrate.     

Multiple molecular forms of phosphorylase phosphatase associated with particulate glycogen and extracted from the cytosol of dog liver.

The glycogen pellet of dog liver extracts contains a phosphorylase phosphatase which has characteristics different from those of the phosphatases extracted from the cytosol. The phosphatase associated with glycogen is characterized by a M, of 51,000, a half maximal inhibition at 0.3 mM ATP (Hill coefficient : 2) and a Ki for Mg2+ of 1 mM. Treatment with urea or mercaptoethanol of the phosphatase associated with glycogen does not influence the activity, the Mr or the half maximal inhibition by ATP, but a decrease of the Hill coefficient for ATP is observed. A similar treatment of the phosphatases extracted from the high speed supernatant results in a decrease of the Mr of the spontaneously active form from 215,000 to 43,000, without an effect on the Ki for ATP (7 micronM), but accompanied by an increase in activity. The ATP-Mg dependent form of the phosphatase from the high speed supernatant (Mr : 138,000 ; Ka for ATP in the presence of 0.1 mM Mg2+ : 0.3 micronM), is denatured by urea or mercaptoethanol. The phosphatase associated with particulate glycogen cannot be found in the supernatant, nor the phosphorylase phosphatases present in the supernatant in the glycogen pellet. When all the glycogen is mobilized (starvation, glucagon) the phosphatase specifically associated with glycogen cannot be found as such in the cytosol. No activation of synthase beta can be detected neither with the phosphatases extracted from the cytosol nor with the enzyme released from the glycogen pellet.     

Activation mechanism of insect fat body phosphorylase by cold Phosphorylase kinase,phosphatase and ATP level

Fat body glycogen phosphorylase of overwintering insects is considerably activated by cold; two enzymes, phosphorylase kinase and phosphatase, may be involved in this activation. In this paper, we demonstrate the mechanism by which phosphorylase is activated by cold. Initially, a latent phosphorylase phosphatase was stimulated by freeze-thawing with 67% saturated ammonium sulphate and mercaptoethanol, enabling partial purification and characterization of this enzyme. Unlike the phosphorylase kinase studied previously, the phosphatase did not function at 0°C. There was no appreciable activation of phosphorylase at 25°C if the phosphatase was present, whereas the phosphorylase was greatly activated at 0°C even in the presence of phosphatase. It is suggested that ATP regulates phosphorylase activity through its effect on kinase and phosphatase. The activation of fat body phosphorylase that naturally occurs in overwintering insects is also discussed.     

Protein phosphatases of the guinea-pig parotid gland

The nature of protein phosphatases of the guinea-pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P-labelled substrate proteins (phosphorylase a, histone H2B, casein, and the alpha and beta subunits of phosphorylase kinase), (b) their behaviour during ion-exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2-mercaptoethanol. The following results were obtained. 1. The 'cytosol' (100,000 X g supernatant) contains protein phosphatases of the types 1, 2A and 2B. 2. On the basis of inhibition with inhibitor 2 (1.2 micrograms/ml) the 'cytosolic' phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A. 3. In the cytosol about 80-90% of the protein phosphatases 1 and 2A exist in an inactive state. 4. A 5-10-fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa. 5. Microsome-associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X-100) or by 0.8 M NaCl. 6. Activity measurements in the presence of inhibitor 2 (1.2 micrograms/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected. 7. The 'microsomal' protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the 'cytosolic' protein phosphatases (about 260 kDa). 8. After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol-treated cytosolic protein phosphatases. 9. Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.     

Phosphoprotein phosphatase associated with rat liver plasma membrane. Properties of phosphorylase phosphatase and phosphohistone phosphatase

Plasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P]phosphorylase a or [32P]phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality. At concentrations as low as 0.1-1.0 mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or Pi. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5 M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl-and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42,000 and 32,000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.     

Native and latent forms of liver phosphorylase phosphatase. The non-identity of native phosphorylase phosphatase and synthase phosphatase.

The directly measurable (native) phosphorylase phosphatase present in a fresh mouse liver extract is bound to particulate glycogen and is not inhibited by heat-stable inhibitors. Treatment of the extract with trypsin or ethanol at room temperature caused a more than 10-fold increase in phosphorylase phosphatase activity. This increased activity stems from the activation of completely inactive (latent) enzyme, the major part of which is present in the high-speed supernatant. The trypsin-revealed activity can be completely blocked by heat-stable inhibitors. Treatment of the animal with glucocorticoids increases, and fasting decreases the activity of the native phosphorylase phosphatase. The level of latent enzyme, however, is unaffected by these treatments. The major portion of synthase phosphatase in the fresh liver extract is bound to glycogen. This enzyme is inhibited by the heat-stable inhibitor-2 and inactivated by trypsin or ethanol as well as by several treatments that have little effect on phosphorylase phosphatase. Upon DEAE-cellulose chromatography at 0 degrees C of a fresh liver extract, phosphorylase phosphatase and synthase phosphatase were resolved as separate, single peaks. If the preparation was not kept at 0 degrees C during the entire procedure, two peaks of each enzyme were observed. Under these conditions the first peak of phosphorylase phosphatase and of synthase phosphatase coincided. From these findings it is concluded that synthase phosphatase and phosphorylase phosphatase, in their native form, are distinct enzymes.     

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.     

The protein phosphatases of Drosophila melanogaster and their inhibitors

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-1, termed protein phosphatase-1I, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-1I. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila.     

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases.

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.     

Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin-sepharose

Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. The latent enzyme had an absolute polycation (histone H1, polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted in the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontaneously active phosphatases were differentiated according to their abilities to dephosphorylate the alpha and the beta subunits of phosphorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-1 phosphatases, respectively.     

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.     

A protein inhibitor of rabbit liver phosphorylase phosphatase.

Extracts of rabbit liver contain a heat-stable, non-dialysable inhibitor of phosphorylase phosphatase. The inhibitory activity is destroyed by trypsin treatment or by ethanol precipitation. The kinetics of the inhibition are non-competitive with respect to phosphorylase a. The inhibitory activity is polydisperse on gel permeation chromatography. The mechanism of the inhibition is due to a direct interaction of the protein inhibitor with the enzyme.     

The release of collagenase as an inactive proenzyme by bone explants in culture

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme-inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a ;procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.     

Purification and properties of a protein inhibitor that inhibits phosphatase 2A activity when hydroxymethylglutaryl coenzyme A reductase is the substrate

A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activity has been identified and purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass was 20,000 Da. The protein lost its inhibitory properties when incubated with trypsin or treated with ethanol. The inhibitor protein does not inhibit type 1 phosphatase when either phosphorylase or hydroxymethylglutaryl-CoA reductase is the substrate. In contrast, this protein inhibitor inhibits the rat liver type 2A phosphatase activity when hydroxymethylglutaryl-CoA reductase is the substrate but not when phosphorylase a is the substrate. The inhibitor protein is not activated by incubation with ATP and cyclic AMP-dependent protein kinase and it is not phosphorylated by glycogen synthase kinase-3. These results, together with those of the kinetic experiments, suggest that the reductase phosphatase inhibitor is distinct from protein phosphatase inhibitor-1 and inhibitor-2.     

Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.     

Evidence for a latent form of protein phosphatase 1 associated with cardiac myofibrils

Detergent-purified myofibrils from bovine heart contained very little spontaneously active protein phosphatase 1 activity. Phosphatase 1, extracted from the myofibrils by freeze-thawing in the presence of 500 mM KCl, was markedly activated by cobalt/trypsin treatment. Myofibril phosphatase 1 was separated from phosphatase 2A by chromatography on heparin-Sepharose. The phosphatase 1 was isolated in a latent form. Pretreatment with trypsin released free catalytic subunit and increased activity about 25-fold. Addition of cobalt with the trypsin increased activity another 2-fold. The latent myofibril phosphatase 1 did not appear to be the same as previously characterized forms of protein phosphatase 1. We suggest that cardiac myofibril phosphatase 1 contains a unique inhibitory subunit which directs the enzyme to the myofibril and regulates the dephosphorylation of myofibril phosphoproteins.     

Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.     

Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human keratinocytes.

Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.