scholarly journals Decreased activity and impaired hormonal control of protein phosphatases in rat livers with a deficiency of phosphorylase kinase

1989 ◽  
Vol 264 (2) ◽  
pp. 429-436 ◽  
Author(s):  
B Toth ◽  
M Bollen ◽  
W Stalmans
Keyword(s):  
Phosphatase Activity ◽  
Wistar Rats ◽  
Glycogen Synthase ◽  
Protein Phosphatases ◽  
Hormonal Control ◽  
Lower Amount ◽  
Phosphorylase Kinase ◽  
Specific Substrate ◽  
Liver Cytosol

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.

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 Identification of high levels of type 1 and type 2A protein phosphatases in higher plants

1989 ◽  
Vol 262 (1) ◽  
pp. 335-339 ◽  
Author(s):  
C MacKintosh ◽  
P Cohen
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Higher Plants ◽  
Protein Phosphatases ◽  
Phosphorylase Kinase ◽  
Rabbit Muscle ◽  
Plant Type ◽  
Mammalian Tissues ◽  
Type 1 Phosphatase

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

scholarly journals The effect of streptozotocin-induced diabetes and of insulin supplementation on glycogen metabolism in rat liver

1977 ◽  
Vol 168 (3) ◽  
pp. 541-548 ◽  
Author(s):  
R L Khandelwal ◽  
S M Zinman ◽  
E J Zebrowski
Keyword(s):  
Phosphatase Activity ◽  
Insulin Treatment ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Diabetic Rats ◽  
Phosphorylase Kinase ◽  
Metabolizing Enzymes ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Streptozotocin Induced Diabetes

The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.

scholarly journals Discovery of a protein phosphatase activity encoded in the genome of bacteriophage λ. Probable identity with open reading frame 221

1989 ◽  
Vol 260 (3) ◽  
pp. 931-934 ◽  
Author(s):  
P T W Cohen ◽  
P Cohen
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Sequence Similarity ◽  
Protein Phosphatases ◽  
Specific Protein ◽  
Open Reading Frame ◽  
Phage Lambda ◽  
Reading Frame

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Induction of hepatic glycogenesis in the fetal rat

1989 ◽  
Vol 256 (1) ◽  
pp. E49-E54 ◽  
Author(s):  
P. A. Gruppuso ◽  
D. L. Brautigan
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Hepatic Glycogen ◽  
Phosphatase Activities ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

We have performed an in vivo study to test the hypothesis that induction of fetal hepatic glycogenesis is stimulated by insulin and involves activation of protein phosphatase type-1. Control animals and the following two experimental groups were studied: maternal fasting for 48 h prior to term and chronic maternal hyperinsulinemia for 5 days prior to term. Maternal fasting led to decreased fetal hepatic glycogen content and fetal growth retardation. In contrast, no decrease in fetal hepatic glycogen content or fetal weight occurred with maternal hyperinsulinemia despite fetal hypoglycemia and fetal hypoinsulinemia. In neither model were fetal hepatic synthase phosphatase or phosphorylase phosphatase activities affected. In control fetuses, the appearance of hepatic glycogen from days 17 to 21 of gestation correlated with induction of glycogen synthase. Phosphorylase phosphatase and synthase phosphatase activities already were present on day 17 of gestation and changed little through term. However, phosphatase catalytic protein reactive with anti-phosphatase type-1 antibodies did increase approximately fivefold from day 18 to 21. In adult animals fasted for 48 h, 50% of hepatic glycogen synthase phosphatase activity was lost, whereas phosphorylase phosphatase activity was stimulated fourfold. The apparent size of protein phosphatase type-1 catalytic subunit as detected by Western immunoblotting was altered by fasting in the adult but not by substrate restriction (maternal fasting) in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin treatment on the activities of phosphoprotein phosphatase and its inhibitors

1980 ◽  
Vol 95 (3) ◽  
pp. 427-432 ◽  
Author(s):  
L. Y. Chang ◽  
L. C. Huang
Keyword(s):  
Skeletal Muscle ◽  
Phosphatase Activity ◽  
Glycogen Synthase ◽  
Rat Skeletal Muscle ◽  
Phosphoprotein Phosphatase ◽  
Heat Stable ◽  
Muscle Extract ◽  
Wide Range ◽  
Heat Stable Protein

Abstract. An increase in glycogen synthase phosphatase (phosphoprotein phosphatase) activity was observed in the rat skeletal muscle extract following insulin administration. The phosphoprotein phosphatase activity present in the muscle extract from insulin treated rats was observed to remain elevated after the extract had been subjected to a molecular sieve chromatography. These results indicate that the stimulatory effets of insulin is due to modification of phosphatase itself or some macromolecular weight modifiers. The heat-stable protein inhibitors of the phosphoprotein phosphatase were isolated from skeletal muscle of insulin treated and control rats and their inhibitory potencies were compared over a wide range of protein concentrations. The inhibitory potency in the insulin treated rat skeletal muscle was found to be significantly less than that in the control muscle. Since type-1 inhibitor is well-known to be active only after being phosphorylated by cyclic AMP-dependent protein kinase, we suggest that the observed change in phosphoprotein phosphatase inhibitor potency is most likely mediated by an alteration in the phosphorylation state of type-1 inhibitor.

scholarly journals Glucose-induced glycogenesis in the liver involves the glucose-6-phosphate-dependent dephosphorylation of glycogen synthase

1997 ◽  
Vol 322 (3) ◽  
pp. 745-750 ◽  
Author(s):  
Joan CADEFAU ◽  
Mathieu BOLLEN ◽  
Willy STALMANS
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Concentration Addition ◽  
Strong Inhibitory Effect ◽  
Glucose Derivatives

Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-α-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1–0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.

scholarly journals Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics

1988 ◽  
Vol 256 (1) ◽  
pp. 283-290 ◽  
Author(s):  
C Bialojan ◽  
A Takai
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Marine Sponge ◽  
Protein Phosphatases ◽  
Kinetic Studies ◽  
High Specificity ◽  
Inhibitory Effect ◽  
Acid Type ◽  
Nitrophenyl Phosphate

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.

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