Rabbit muscle phosphorylase phosphatase. 1. Purification and chemical properties

Gratecos, D.; Detwiler, Thomas C.; Hurd, Susan; Fischer, Edmond H.

doi:10.1021/bi00641a009

Cited by 93 publications

(34 citation statements)

References 36 publications

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“…The enzyme was eluted with 6 M urea and many contaminating proteins precipitated on subsequent dialysis at low ionic strength. Since protein phosphatase-1 accounts for virtually all the phosphorylase phosphatase activity in the protein-glycogen complex (see Results and [22]), the enzyme isolated by Gratecos et al [42] must be this enzyme. However, they stated that the purified enzyme was unable to dephosphorylate phosphorylase kinase (either subunit), glycogen synthase or histones.…”

Section: Comparison With the Work Of Kobayashi And Kuto [3kjmentioning

confidence: 95%

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The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

1983

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The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-I were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-I, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 232, 255-2611 were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors.Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCI), 2A1 (0.2 M NaCI) and 2Az (0.28 M NaCI) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-M,,,,,, catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2Az to more active 34000-Mr~,,,~ catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2Az possessed identical substrate specificities, preferentially dephosphorylated the r-subunit of phosphorylase kinase, were unaffected by inhibitor-I and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2Al and 2Az were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP.Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98 000. Protein phosphatase-2B was completely inhibited by 100 pM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B.Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2Al. These activities were completely resolved by gel filtration, since the Mr(,,,) of protein phosphatase-2C was 46000.Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg . ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2Al. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2.Most, if not all, of the protein phosphatases described in the literature as enzymes active on the phosphorylated proteins of glycogen metabolism can now b...

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Section: Comparison With the Work Of Kobayashi And Kuto [3kjmentioning

confidence: 95%

“…Gratecos et al [42] purified phosphorylase phosphatase from the protein-glycogen complex by chromatography on poly(1y-sine)-Sepharose. The enzyme was eluted with 6 M urea and many contaminating proteins precipitated on subsequent dialysis at low ionic strength.…”

Section: Comparison With the Work Of Kobayashi And Kuto [3kjmentioning

confidence: 99%

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

1983

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“…The idea that PP1 may be a useful target for future therapeutic purposes has recently been enhanced by the observations that targeting of PP1 by a herpesvirus protein is part of the viral mechanism for overcoming host defenses (17) and by the observation that HOX11, which is oncogenic in human T-cell leukemia, interacts with PP1 and PP2A (6).…”

Section: Table I Alignment Of Peptide Sequences That Bind To Pp1mentioning

confidence: 99%

A Protein Phosphatase-1-binding Motif Identified by the Panning of a Random Peptide Display Library

Zhao

Lee

1997

Journal of Biological Chemistry

133

125

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An unusually large number of regulatory or targeting proteins that bind to the catalytic subunit of protein phosphatase-1 have been recently reported. This can be explained by their possession of a common protein motif that interacts with a binding site on protein phosphatase-1. The existence of such a motif was established by the panning of a random peptide library in which peptide sequences are displayed on the Escherichia coli bacterial flagellin protein for bacteria that bound to protein phosphatase-1. There were 79 isolates containing 46 unique sequences with the conserved motif VXF or VXW, where X was most frequently His or Arg. In addition, this sequence was commonly preceded by 2-5 basic residues and followed by 1 acidic residue. This study demonstrates that binding to protein phosphatase-1 can be conferred to a protein by the presentation of a peptide motif on a surface loop. This binding motif is found in a number of protein phosphatase-1-binding proteins.Protein phosphatase-1, originally studied in the context of glycogen metabolism as the enzyme that converts phosphorylase a to phosphorylase b (1), has been implicated in the regulation of a number of important cellular processes (for reviews, see Refs. 2 and 3). Biochemical studies have revealed that it consists of a catalytic subunit (4 -7) of 37 kDa (PP1) 1 that forms a number of heterodimeric enzymes with different subunits, which include a glycogen-binding subunit, a myosin-binding subunit (8), and inhibitor-2 (9). These subunits function to target the catalytic subunit to the subcellular or molecular proximity of its substrates and may serve to provide regulation of activity as well as modulation of substrate specificity (8). In addition, PP1 is regulated by several inhibitory proteins; these include inhibitor-1 (10), DARPP-32 (10), inhibitor-2 and NIPP (a nuclear inhibitory protein) (11). The use of the yeast twohybrid system has led to the discovery of a surprisingly large number of PP1-binding proteins. Mammalian PP1-binding proteins include the retinoblastoma gene product (12), HSP78 (13), p53BP2 (14), splicing factor PSF (15), ribosomal protein L5 (16), herpesvirus ␥ 1 34.5 protein (17), and HOX11 (18). In yeast, over a dozen genes that encode PP1-binding proteins have been identified, based largely on the use of a two-hybrid screen. These include genes that are variously required for control of glycogen metabolism, glucose repression, meiosis and/or sporulation, and mitotic cell cycle regulation (19 -21). Thus, PP1 is unusual in that this single enzyme is involved in the regulation of a number of diverse cellular processes.Few, if any, of the PP1 proteins share any major sequence identity, although examination of different glycogen-binding subunits has revealed the presence of two small regions of sequence similarity that is shared between several glycogenbinding proteins (20,(22)(23)(24)(25). The unusually large number of PP1-binding proteins that have been described suggests either that PP1 contains a motif that is recognized by a common...

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“…The activities toward phosphohistone and the phosphocasein were found to co-purify with that toward phosphorylase a throughout the purification procedures, indicating that the enzyme has a broad specificity. The nonspecific nature of the enzyme is similar to that of phosphoprotein phosphatase of M, in the range 30000-35000 purified from liver [25,36], cardiac [19,37] and skeletal muscle [38,39]. These low-molecular-weight forms of the enzyme have been described to be active toward a variety of phosphoproteins, including phosphorylase a, glycogen synthetase D, phosphorylase kinase, pyruvate kinase, hormone-sensitive lipase, phos-phorylated regulatory subunit of type I1 cyclic-AMPdependent protein kinase, troponin, histone and casein.…”

Section: Discussionmentioning

confidence: 86%

“…The observation that prolonged incubation of phosphorylase phosphatase with high Pi concentration (0.5 M [45]) can convert the enzyme into a metal-iondependent form may reflect the fact that Pi can complex and remove the essential divalent cation(s) from the enzyme and its chelating ability is much poorer than that of pyrophosphoryl compounds. Gratecos et al [38] have reported that a skeletal muscle phosphorylase phosphatase (MI 33 000) displays a strong tendency to aggregate with loss of enzymic activity. In the present studies, we found that PPi-inactiveted or ATP-inactivated enzyme has the same molecular size as that of the untreated enzyme.…”

Section: Discussionmentioning

confidence: 99%

Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of M_r 35000 from Bovine Adrenal Cortex

1979

European Journal of Biochemistry

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A metal-ion-independent, nonspecific phosphoprotein phosphatase (MI = 35 000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ > Mn2+ > Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ > Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.The covalent modification of enzymes and regulatory proteins through a cycle of phosphorylation and dephosphorylation reactions is an important control mechanism involved in the regulation of many biological processes [l -51. Evidence indicates that the effects of adrenocorticotropic hormone on steroidogenesis [6-91 and on glycogen phosphorylase [lo, 111 are mediated by cyclic AMP and protein phosphorylation. In the past years, several reports have appeared relative to purification, specificity, and regulation of adrenal cortex phosphoprotein phosphatase [lo-141. In spite of these efforts, a homogeneous preparation of the enzyme has not yet been obtained. In addition, the basic catalytic properties and mechanism of regulation of the adrenal cortex phosphorylase phosphatase remains unclear.

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Rabbit muscle phosphorylase phosphatase. 1. Purification and chemical properties

Cited by 93 publications

References 36 publications

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

A Protein Phosphatase-1-binding Motif Identified by the Panning of a Random Peptide Display Library

Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of M_r 35000 from Bovine Adrenal Cortex

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Rabbit muscle phosphorylase phosphatase. 1. Purification and chemical properties

Cited by 93 publications

References 36 publications

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

A Protein Phosphatase-1-binding Motif Identified by the Panning of a Random Peptide Display Library

Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of Mr 35000 from Bovine Adrenal Cortex

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Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of M_r 35000 from Bovine Adrenal Cortex