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.

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 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.

Effects of Serine/Threonine Protein Phosphatases on Ion Channels in Excitable Membranes

2000 ◽  
Vol 80 (1) ◽  
pp. 173-210 ◽  
Author(s):  
Stefan Herzig ◽  
Joachim Neumann
Keyword(s):  
Ion Channels ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Direct Interaction ◽  
Specific Protein ◽  
Biochemical Properties ◽  
Future Research ◽  
Regulatory Subunits ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors

This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues. Particular focus is given to developments of the past decade. Most of the electrophysiological experiments have been performed with protein phosphatase inhibitors. Therefore, a synopsis is required incorporating issues from biochemistry, pharmacology, and electrophysiology. First, we summarize the structural and biochemical properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3–7) catalytic subunits and their regulatory subunits. Then the available pharmacological tools (protein inhibitors, nonprotein inhibitors, and activators) are introduced. The use of these inhibitors is discussed based on their biochemical selectivity and a number of methodological caveats. The next section reviews the effects of these tools on various classes of ion channels (i.e., voltage-gated Ca2+ and Na+ channels, various K+ channels, ligand-gated channels, and anion channels). We delineate in which cases a direct interaction between a protein phosphatase and a given channel has been proven and where a more complex regulation is likely involved. Finally, we present ideas for future research and possible pathophysiological implications.

scholarly journals N-Acetyl-β-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1

1997 ◽  
Vol 328 (2) ◽  
pp. 695-700 ◽  
Author(s):  
Mary BOARD
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Protein Phosphatase 1 ◽  
P Concentration ◽  
Glucose Analogue ◽  
Stimulation Of

Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-β-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 μM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 μM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 μM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented.

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats

2001 ◽  
Vol 360 (2) ◽  
pp. 449-459 ◽  
Author(s):  
Gareth J. BROWNE ◽  
Mirela DELIBEGOVIC ◽  
Stefaan KEPPENS ◽  
Willy STALMANS ◽  
Patricia T. W. COHEN
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Diabetic Rats ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Hepatic Glycogen ◽  
Insulin Dependent ◽  
Insulin Dependent Diabetic

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, GL, R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, GL–PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and GL complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and GL were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6–PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and GL and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for GL. The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of GL–PP1c, suggesting that R5–PP1c may function as a hepatic phosphorylase phosphatase, whereas GL–PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and GL

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 Identification of a Pneumococcal Glycosidase That Modifies O-Linked Glycans

2009 ◽  
Vol 77 (4) ◽  
pp. 1389-1396 ◽  
Author(s):  
Carolyn Marion ◽  
Dominique H. Limoli ◽  
Gregory S. Bobulsky ◽  
Jessica L. Abraham ◽  
Amanda M. Burnaugh ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Bifidobacterium Longum ◽  
Airway Epithelial Cells ◽  
Open Reading Frame ◽  
Glycan Structure ◽  
Dependent Manner ◽  
Upper Respiratory Tract ◽  
Reading Frame ◽  
Human Airway ◽  
Published Evidence

ABSTRACT Colonization of the airway by Streptococcus pneumoniae is typically asymptomatic; however, progression of bacteria beyond the oronasopharynx can cause diseases including otitis media and pneumonia. The mechanisms by which S. pneumoniae establishes and maintains colonization remain poorly understood. Both N-linked and O-linked glycans are abundant in the airway. Our previous research demonstrated that S. pneumoniae can sequentially deglycosylate N-linked glycans and suggested that this modification of sugar structures may aid in colonization. There is published evidence that S. pneumoniae expresses a secreted O-glycosidase that cleaves galactose β1-3 N-acetylgalactosamine (Galβ1-3GalNAc) from core-1 O-linked glycans; however, the biological function of this enzyme has not previously been determined. We established that the activity is not secreted but is instead surface associated in a sortase-dependent manner. Genome analysis revealed an open reading frame predicted to encode a sortase-dependent surface protein with sequence similarity to the O-glycosidase of Bifidobacterium longum. Deletion of this pneumococcal open reading frame confirmed that this gene encodes an O-glycosidase. Experiments using a model glycoconjugate demonstrated that this O-glycosidase, together with the neuraminidase NanA, is required for S. pneumoniae to cleave sialylated core-1 O-linked glycans. The ability of the O-glycosidase mutant to cleave this glycan structure was restored by both genetic complementation and the addition of O-glycosidase. The mutant showed a reduction in adherence to human airway epithelial cells and a significantly decreased ability to colonize the upper respiratory tract, suggesting that cleavage of core-1 O-linked glycans enhances the ability of S. pneumoniae to colonize the human airway.

scholarly journals Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle

1986 ◽  
Vol 239 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J Goris ◽  
E Waelkens ◽  
W Merlevede
Keyword(s):  
Skeletal Muscle ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Activity Ratio ◽  
Protein Phosphatases ◽  
Histone H1 ◽  
Rabbit Skeletal Muscle ◽  
Significant Activity ◽  
Purification Step ◽  
Apparent Homogeneity

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.

Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cells

2009 ◽  
Vol 25 (3) ◽  
pp. 735-744 ◽  
Author(s):  
Yune Z. Kunes ◽  
Wendy R. Gion ◽  
Emma Fung ◽  
Jochen G. Salfeld ◽  
Rong-Rong Zhu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Open Reading Frame ◽  
Single Open Reading Frame ◽  
Reading Frame ◽  
Polyprotein Processing
Sign in / Sign up

Export Citation Format

Share Document