scholarly journals Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver

1985 ◽  
Vol 225 (3) ◽  
pp. 665-670 ◽  
Author(s):  
G Mieskes ◽  
H D Söling
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Liver Protein ◽  
Key Enzyme ◽  
Phosphatase 2A

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.

scholarly journals Dephosphorylation of 1d-myo-inositol 1,4-bisphosphate in rat liver

1988 ◽  
Vol 254 (3) ◽  
pp. 655-660 ◽  
Author(s):  
A J Morris ◽  
D J Storey ◽  
C P Downes ◽  
R H Michell
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Substrate Concentration ◽  
Gel Filtration ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Inositol Monophosphatase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.

scholarly journals Insulin-receptor phosphotyrosyl-protein phosphatases

1988 ◽  
Vol 256 (3) ◽  
pp. 893-902 ◽  
Author(s):  
M J King ◽  
G J Sale
Keyword(s):  
Insulin Receptor ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Egf Receptor ◽  
Soluble Fraction ◽  
Protein Phosphatases ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Dependent Protein

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.

scholarly journals Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 423-433 ◽  
Author(s):  
J F Antoniw ◽  
H G Nimmo ◽  
S J Yeaman ◽  
P Cowen
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Substrate Specificities ◽  
Phosphatase Activities

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

scholarly journals Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

1993 ◽  
Vol 4 (7) ◽  
pp. 669-677 ◽  
Author(s):  
P Ferrigno ◽  
T A Langan ◽  
P Cohen
Keyword(s):  
Rat Liver ◽  
Protein Phosphatase ◽  
Human Fibroblasts ◽  
High Mobility ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Mobility Group Protein ◽  
Cell Extracts ◽  
Dependent Protein

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.

scholarly journals The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen

1988 ◽  
Vol 250 (3) ◽  
pp. 659-663 ◽  
Author(s):  
M Bollen ◽  
W Stalmans
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Soluble Fraction ◽  
Glycogen Synthase ◽  
Gel Filtration ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Phosphatase Activities ◽  
Glycogen Particles

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).

Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase

1970 ◽  
Vol 48 (7) ◽  
pp. 799-804 ◽  
Author(s):  
C. Hétu ◽  
R. Gianetto
Keyword(s):  
Rat Liver ◽  
Molecular Sieve ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Filtration Step ◽  
Hydrolysis Of ◽  
Menten Constant

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.

scholarly journals Activity of protein phosphatases against initiation factor-2 and elongation factor-2

1990 ◽  
Vol 272 (1) ◽  
pp. 175-180 ◽  
Author(s):  
N T Redpath ◽  
C G Proud
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Protein Phosphatase 2A ◽  
Elongation Factor ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Elongation Factor 2 ◽  
Initiation Factor 2 ◽  
Phosphatase 2A

The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

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