Rabbit muscle phosphorylase phosphatase. 2. Kinetic properties and behavior in glycogen particles

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

doi:10.1021/bi00641a010

Cited by 52 publications

(21 citation statements)

References 14 publications

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“…The enzyme can be partially purified by procedures similar to those described for the preparation of the corresponding enzymes from animal tissues (2)(3)(4)(5)(6)(7)(8). Ethanol precipitation at room temperature apparently is an important step in the purification.…”

Section: Discussionmentioning

confidence: 99%

“…Evidently, phosphoprotein phosphatases in general do not catalyze the hydrolysis of nonprotein phosphoesters (5,6,23). Second, the estimated mol wt of the soybean enzyme is within the range of 30,000 to 40,000 obtained for the corresponding highly purified enzymes from animal tissues (5,6,10,11,16,23). Third, lack of specificity for phosphoprotein substrate is generally observed for phosphoprotein phosphatases, even those purified to apparent homogeneity from animal tissues (10,18).…”

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confidence: 99%

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Phosphoprotein Phosphatase of Soybean Hypocotyls

Lin

Mori²,

Key³

1980

Plant Physiol.

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A soybean histone-type protein kinase was used to prepare 32P-labeled histone Hi as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.13.16) phorylation and dephosphorylation has been suggested to be an important mechanism in the control of metabolic processes in higher plants (12,25). Randall and his colleagues (21) have shown that phosphorylation of the pyruvate dehydrogenase complex from broccoli mitochondria results in inactivation of the enzyme. Additionally, the nuclear (17), ribosomal (20, 24) and membrane proteins (1) of higher plants are also subject to modification by phosphorylation, although the biological significance of the phosphorylation is unknown. The phosphorylation of these proteins apparently is catalyzed by several protein kinases, including histone-type and casein (or phosvitin)-type enzymes (9,12,25). Although much work has been carried out on phosphorylation of proteins and demonstration of the activities of protein kinases, little attention has been paid to phosphoprotein phosphatase (EC 3.1.3.16) which catalyzes the protein dephosphorylation reaction. There apparently has been no characterization of the phosphatase from higher plants, except a brief mention of the presence of phosphohistone phosphatase activity in pea seedlings (16). Here, we describe the purification and properties of a phosphoprotein phosphatase from soybean hypocotyls. The enzyme is distinct from the phosphatases which catalyze the hydrolysis ofnonprotein low mol wt phosphoesters. Additionally, several lines of evidence indicate that the soybean enzyme is a nonspecific phosphoprotein phosphatase acting on phosphorylated histones, protamine, and, possibly, phosphorylated casein and phosvitin. MATERIALS AND METHODSUnless otherwise stated, the chemicals and biochemical compounds used in this study were obtained from the sources as indicated in the previous paper (12). [y-32PJATP was prepared according to the method of Schendel and Wells (22). All other chemicals were of reagent grade. Soybean seeds (Glycine max var. Wayne) were germinated in the dark and the mature hypocotyls were harvested as described (12).Preparation of 32P-labeled and Nonradioactive Phosphohistone Proteins. Lysine-rich histone HI and other histone species (H2A, H2B, H3, and H4) were purified from the total calfthymus histone mixture (Sigma II or IIA) by column chromatographies on BioGel P-60 and P-10 (12, 14). A histone-type protein kinase with high specificity for histone HI was partially purified from soybean hypocotyls as described (12); the kinase was used to phosphorylate histone H1. For the preparation of 32P-labeled histone species other than histone H1, a cAMP-dependent histone kinase from rat liver cytosol (4) was used. The liver kinase was partially purified by ammonium sulfate fractionation and DEAE-Sephadex A-25 column chromatography.To prepare a large quantity of 32P-labeled histone H1, the reaction mixture in a total volume of 10 ml contained 20 mM Tris-HCI (pH 8.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Phosphoprotein Phosphatase of Soybean Hypocotyls

Lin

Mori²,

Key³

1980

Plant Physiol.

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“…Since substrate-directed factors can either enhance or impede dephosphorylation of a protein (27,28), we tested whether dephosphorylation of PDE5 by phosphoprotein phosphatases is affected by a ligand-induced change in conformation of the enzyme. PDE5 was phosphorylated quantitatively (~0.7 -1 mol phosphate per PDE5 monomer) in the absence of cGMP as described under Materials and Methods.…”

Section: Ligands Do Not Regulate Dephosphorylation Of Pde5mentioning

confidence: 99%

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil

Bessay

Zoraghi²,

Blount³

et al. 2007

Front Biosci

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Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

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“…Evidence has also been accumulated that the phosphoprotein phosphatase may be controlled by both covalent and non-covalent modification of the substrate. Namely, AMP, ATP [9,10], glucose 6-phosphate [l 11, polyamine hydrochlorides [12] and other salts, such as NaCl and magnesium acetate [12,13], confer significant changes in enzymatic activity by acting on the substrates in an allosteric manner. Another form of phosphatase control is through changes in the degree of phosphorylation of the substrate.…”

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confidence: 99%

The Control of Phosphoprotein Phosphatase by the Second‐Site Phosphorylation of a Substrate

Usui

Nishimura

Imazu

et al. 1979

European Journal of Biochemistry

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The phosphorylation of Ser-32, in addition to Ser-36 of H2B histone, stimulated the rate of Pi release from Ser-36 by the small form ( M , 31 000) of pig heart phosphoprotein phosphatase both in the absence and presence of 50 mM magnesium acetate. By phosphorylation at Ser-32, the K, value for Ser-36 phosphate in H2B histone was increased from 0.38 pM to 1.16 pM in the absence of magnesium acetate, but not significantly changed (from 37.4 pM to 26.2 pM) in the presence of magnesium acetate. With the large form ( M , 224000) of the phosphoprotein phosphatase, however, the phosphorylation at Ser-32 suppressed the rate of Pi release from Ser-36 both in the absence and presence of magnesium acetate. The K , value of the large form for Ser-36 phosphate in H2B histone was nevertheless increased by phosphorylation at Ser-32, from 1.2 pM to 5.3 pM in the presence of magnesium acetate, but not changed (from 0.26 pM to 0.23 pM) in the absence of magnesium acetate.Recent investigations [l-81 indicate that a single phosphoprotein phosphatase catalyzes the dephosphorylations of phosphorylase a, glycogen synthetase D, phosphorylase h kinase and other proteins phosphorylated by cyclic-AMP-dependent (adenosine-3' : 5'-monophosphate-dependent) protein kinase. Evidence has also been accumulated that the phosphoprotein phosphatase may be controlled by both covalent and non-covalent modification of the substrate. Namely, AMP, ATP [9,10], glucose 6-phosphate [l 11, polyamine hydrochlorides [12] and other salts, such as NaCl and magnesium acetate [12,13], confer significant changes in enzymatic activity by acting on the substrates in an allosteric manner. Another form of phosphatase control is through changes in the degree of phosphorylation of the substrate. Cohen and Antoniw [14] have reported that the phosphorylation of M subunits of muscle phosphorylase kinase increases the susceptibility of its subunits to the phosphoprotein phosphatase. Hutson et al. [15] the conversion of glycogen synthetase D to I by the phosphatase is stimulated by the second-site phosphorylation on the same subunit of the synthetase by either cyclic-AMP-dependent protein kinase or independent protein kinase. In a previous paper [8] we described the partial purification of a pig heart phosphoprotein phosphatase (large form, M , 224000) and its conversion to a smaller form (small form, M , 3 1000) by ethanol treatment. These two forms of the phosphoprotein phosphatase preferentially dephosphorylate Ser-36 phosphate of H2B histone which is phosphorylated at both Ser-32 and Ser-36 by cyclic-AMPdependent protein kindse [8].In this paper we will report the effects of the additional phosphorylation of Ser-32 on the dephosphorylation of Ser-36 phosphate in H2B histone. The data will indicate that the activity of enzyme is greatly influenced by the additional phosphorylation, and that the dissociation of the large form of the enzyme to the small form is accompanied by a change in the response of the enzyme to such covalent modification of the substrate. MATERIALS...

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Rabbit muscle phosphorylase phosphatase. 2. Kinetic properties and behavior in glycogen particles

Cited by 52 publications

References 14 publications

Phosphoprotein Phosphatase of Soybean Hypocotyls

Phosphoprotein Phosphatase of Soybean Hypocotyls

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil

The Control of Phosphoprotein Phosphatase by the Second‐Site Phosphorylation of a Substrate

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