Simple, Sensitive Detection of Protein Phosphodiesterase Activity Measure Real-Time Signal Differences in the Picomole Range with the Phosphate Sensor Method
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Calmodulin-Dependent Protein Kinase II–Protein Phosphatase 1 Switch Facilitates Specificity in Postsynaptic Calcium Signaling
An ultrasensitive Ca2؉͞calmodulin-dependent protein kinase II–protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling J. Michael Bradshaw*†‡, Yoshi Kubota*, Tobias Meyer†, and Howard Schulman* Departments of *Neurobiology and †Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305 Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved July 21, 2003 (received for review May 7, 2003) The strength of hippocampal synapses can be persistently in- hence would provide a distinct Ca2ϩ activation region for creased by signals that activate Ca2؉͞calmodulin-dependent pro- CaMKII compared with other Ca2ϩ-activated enzymes at the tein kinase II (CaMKII). This CaMKII-dependent long-term potenti- synapse. In fact, it has been hypothesized that the signaling ation is important for hippocampal learning and memory. In this network controlling CaMKII autophosphorylation is a bistable work we show that CaMKII exhibits an intriguing switch-like type of switch that allows CaMKII to remain autophosphory- activation that likely is important for changes in synaptic strength. lated long after Ca2ϩ returns to a basal level (16). We found that autophosphorylation of CaMKII by itself showed a In this work we demonstrate experimentally that CaMKII 2؉ Ϸ ϩ steep dependence on Ca concentration [Hill coefficient (nH) 5]. responds in a switch-like fashion to Ca2 : CaMKII transitions 2؉ Ϸ However, an even steeper Ca dependence (nH 8) was observed rapidly from little to near-total autophosphorylation over a when autophosphorylation is balanced by the dephosphorylation narrow range of Ca2ϩ. Interestingly, this switch-like response was activity of protein phosphatase 1 (PP1). -
(PDE 1 B 1) Correlates with Brain Regions Having Extensive Dopaminergic Innervation
The Journal of Neuroscience, March 1994, 14(3): 1251-l 261 Expression of a Calmodulin-dependent Phosphodiesterase lsoform (PDE 1 B 1) Correlates with Brain Regions Having Extensive Dopaminergic Innervation Joseph W. Polli and Randall L. Kincaid Section on Immunology, Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852 Cyclic nucleotide-dependent protein phosphorylation plays PDE implies an important physiological role for Ca2+-regu- a central role in neuronal signal transduction. Neurotrans- lated attenuation of CAMP-dependent signaling pathways mitter-elicited increases in cAMP/cGMP brought about by following dopaminergic stimulation. activation of adenylyl and guanylyl cyclases are downre- [Key words: CAMP, cyclase, striatum, dopamine, basal gulated by multiple phosphodiesterase (PDE) enzymes. In ganglia, DARPP-321 brain, the calmodulin (CaM)-dependent isozymes are the major degradative activities and represent a unique point of Cyclic nucleotides, acting as “second messengers”or via direct intersection between the cyclic nucleotide- and calcium effects, regulate a diverse array of neuronal functions, from ion (Ca*+)-mediated second messenger systems. Here we de- channel conductance to gene expression. Hydrolysis of 3’,5’- scribe the distribution of the PDEl Bl (63 kDa) CaM-depen- cyclic nucleotidesto 5’-nucleosidemonophosphates is the major dent PDE in mouse brain. An anti-peptide antiserum to this mechanismfor decreasingintracellular cyclic nucleotide levels. isoform immunoprecipitated -3O-40% of cytosolic PDE ac- This reaction is catalyzed by cyclic nucleotide phosphodiester- tivity, whereas antiserum to PDElA2 (61 kDa isoform) re- ase (PDE) enzymes that constitute a large superfamily (Beavo moved 60-70%, demonstrating that these isoforms are the and Reifsynder, 1990). -
Compartment-Specific Phosphorylation of Phosducin In
Faculty Scholarship 2007 Compartment-specific hoP sphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination Hongman Song Marycharmain Belcastro E. J. Young Maxim Sokolov Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Digital Commons Citation Song, Hongman; Belcastro, Marycharmain; Young, E. J.; and Sokolov, Maxim, "Compartment-specific hospP horylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination" (2007). Faculty Scholarship. 92. https://researchrepository.wvu.edu/faculty_publications/92 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted for inclusion in Faculty Scholarship by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 32, pp. 23613–23621, August 10, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination* Received for publication, March 7, 2007, and in revised form, June 11, 2007 Published, JBC Papers in Press, June 14, 2007, DOI 10.1074/jbc.M701974200 Hongman Song, Marycharmain Belcastro, E. J. Young, and Maxim Sokolov1 From the Departments of Ophthalmology and Biochemistry, West Virginia University School of Medicine and West Virginia University Eye Institute, Morgantown, West Virginia 26506 Phosducin is a major phosphoprotein of rod photoreceptors specific complex with the ␥ subunits of visual heterotrimeric that interacts with the G␥ subunits of heterotrimeric G pro- G protein, transducin (4, 5), and other heterotrimeric G pro- teins in its dephosphorylated state. -
Reduction of Pectinesterase Activity in a Commercial Enzyme Preparation
Journal of the Science of Food and Agriculture J Sci Food Agric 85:1613–1621 (2005) DOI: 10.1002/jsfa.2154 Reduction of pectinesterase activity in a commercial enzyme preparation by pulsed electric fields: comparison of inactivation kinetic models Joaquın´ Giner, Pascal Grouberman, Vicente Gimeno and Olga Martın´ ∗ Department of Food Technology, University of Lleida, CeRTA-UTPV, ETSEA, Avda Alcalde Rovira Roure 191, 25198-Lleida, Spain Abstract: The inactivation of pectinesterase (PE) in a commercial enzyme preparation (CEP) under high intensity pulsed electric fields (HIPEF) was studied. After desalting and water dilution of the raw CEP, samples were exposed to exponentially decay waveform pulses for up to 463 µs at electric field intensities ranging from 19 to 38 kV cm−1. Pulses were applied in monopolar mode. Experimental data were fitted to a first-order kinetic model as well as to models based on Fermi, Hulsheger¨ or Weibull equations to describe PE inactivation kinetics. Characteristic parameters for each model were calculated. Relationships between some of the parameters and process variables were obtained. The Weibull model yielded the best accuracy factor. The relationship between residual PE and input of electrical energy density was found to be that of exponential decay. 2005 Society of Chemical Industry Keywords: pulsed electric fields; kinetics; pectinesterase; model; inactivation INTRODUCTION It has become customary to use CEPs in fruit and Pectinesterase (PE; EC 3.1.1.11) is a pectic enzyme vegetable juice technology. Depending -
Regulation of Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase (PDE1): Review
95-105 5/6/06 13:44 Page 95 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 18: 95-105, 2006 95 Regulation of calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1): Review RAJENDRA K. SHARMA, SHANKAR B. DAS, ASHAKUMARY LAKSHMIKUTTYAMMA, PONNIAH SELVAKUMAR and ANURAAG SHRIVASTAV Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Cancer Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon SK S7N 4H4, Canada Received January 16, 2006; Accepted March 13, 2006 Abstract. The response of living cells to change in cell 6. Differential inhibition of PDE1 isozymes and its environment depends on the action of second messenger therapeutic applications molecules. The two second messenger molecules cAMP and 7. Role of proteolysis in regulating PDE1A2 Ca2+ regulate a large number of eukaryotic cellular events. 8. Role of PDE1A1 in ischemic-reperfused heart Calmodulin-stimulated cyclic nucleotide phosphodiesterase 9. Conclusion (PDE1) is one of the key enzymes involved in the complex interaction between cAMP and Ca2+ second messenger systems. Some PDE1 isozymes have similar kinetic and 1. Introduction immunological properties but are differentially regulated by Ca2+ and calmodulin. Accumulating evidence suggests that the A variety of cellular activities are regulated through mech- activity of PDE1 is selectively regulated by cross-talk between anisms controlling the level of cyclic nucleotides. These Ca2+ and cAMP signalling pathways. These isozymes are mechanisms include synthesis, degradation, efflux and seque- also further distinguished by various pharmacological agents. stration of cyclic adenosine 3':5'-monophosphate (cAMP) and We have demonstrated a potentially novel regulation of PDE1 cyclic guanosine 3':5'- monophosphate (cGMP) within the by calpain. -
The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic—Facts and Questions
International Journal of Molecular Sciences Review The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic—Facts and Questions Csaba Máthé *, Márta M-Hamvas, Csongor Freytag and Tamás Garda Department of Botany, Faculty of Science and Technology, University of Debrecen, H-4032 Debrecen, Hungary; [email protected] (M.M.-H.); [email protected] (C.F.); [email protected] (T.G.) * Correspondence: [email protected] Abstract: The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP–GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole Citation: Máthé, C.; M-Hamvas, M.; plant development. -
G Protein Regulation of MAPK Networks
Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules. -
Download Product Insert (PDF)
PRODUCT INFORMATION Lysophospholipase D Polyclonal Antibody Item No. 10005375 Overview and Properties Contents: This vial contains 500 µl of peptide affinity-purified antibody. Synonyms: Autotaxin, ENPP2, lysoPLD Immunogen: Peptide from the C-terminal region of rat LysoPLD Species Reactivity: (+) Human, mouse, and rat; other species not tested Form: Liquid Storage: -20°C (as supplied) Stability: ≥1 year Storage Buffer: TBS, pH 7.4, with 50% glycerol, 0.1%l BSA, and 0.02% sodium azide Host: Rabbit Applications: Immunocytochemistry (ICC), Immunohistochemistry (IHC), and Western blot (WB); the recommended starting dilution for ICC is 1:500, 1:80 for IHC, and 1:200 for WB. Other applications were not tested, therefore optimal working concentration/dilution should be determined empirically. Images 1 · · · · · · · 104 kDa · · · · · · · 60 kDa Lane 1: Human cerebella supernatant (40 µg) Immunohistochemistry analysis of formalin-fixed, paraffin-embedded (FFPE) human cerebellum ǎssue aer heat-induced anǎgen retrieval in pH 6.0 citrate buffer. Aer incubaǎon with Lysophospholipase D Polyclonal Anǎbody (Item No. 10005375) at a 1:80 diluǎon, slides were incubated with bioǎnylated secondary anǎbody, followed by alkaline phosphatase-strepavidin and chromogen (DAB). WARNING CAYMAN CHEMICAL THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE. 1180 EAST ELLSWORTH RD SAFETY DATA ANN ARBOR, MI 48108 · USA This material should be considered hazardous until further information becomes available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash thoroughly after handling. Before use, the user must review the complete Safety Data Sheet, which has been sent via email to your institution. PHONE: [800] 364-9897 WARRANTY AND LIMITATION OF REMEDY [734] 971-3335 Buyer agrees to purchase the material subject to Cayman’s Terms and Conditions. -
Protein Phosphatases and Calcium/Calmodulin-Dependent Protein Kinase II-Dependent Synaptic Plasticity
8404 • The Journal of Neuroscience, September 29, 2004 • 24(39):8404–8409 Mini-Review Protein Phosphatases and Calcium/Calmodulin-Dependent Protein Kinase II-Dependent Synaptic Plasticity Roger J. Colbran Department of Molecular Physiology and Biophysics, The Center for Molecular Neuroscience and The Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615 Key words: calcium; calmodulin; learning; localization; LTP; NMDA; phosphatase; protein kinase; postsynaptic density Synaptic plasticity in hippocampal CA1 pyramidal cells requires a of the catalytic (C) subunits (two genes) with structural (A) sub- delicate balance of protein kinase and protein phosphatase activ- units (two genes) and regulatory (B) subunits (four known un- ities. Long-term potentiation (LTP) after intense synaptic stim- related gene families), and these can additionally associate with ulation often results from postsynaptic Ca 2ϩ influx via NMDA- other cellular proteins (Virshup, 2000). Similarly, four PP1 cata- ␣  ␥ ␥ type glutamate receptors and activation of multiple protein lytic subunit isoforms ( , , 1, and 2) differentially interact kinases. In contrast, weaker synaptic stimulation paradigms can with Ͼ40 known regulatory and/or targeting subunits (Ceule- induce long-term depression (LTD) (or depotentiation of previ- mans and Bollen, 2004). Thus, to understand how phosphatases ous LTP) via NMDA receptor-dependent activation of protein modulate processes such as synaptic plasticity, it is critical to serine/threonine phosphatases. Autophosphorylations at Thr 286, identify both the type of phosphatase that dephosphorylates a Thr 305, and Thr 306 regulate the activity and subcellular local- physiologically relevant substrate and the molecular nature of ization of calcium/calmodulin-dependent protein kinase II relevant protein phosphatase complex(es). -
Diagnostic Value of Serum Enzymes-A Review on Laboratory Investigations
Review Article ISSN 2250-0480 VOL 5/ ISSUE 4/OCT 2015 DIAGNOSTIC VALUE OF SERUM ENZYMES-A REVIEW ON LABORATORY INVESTIGATIONS. 1VIDYA SAGAR, M.SC., 2DR. VANDANA BERRY, MD AND DR.ROHIT J. CHAUDHARY, MD 1Vice Principal, Institute of Allied Health Sciences, Christian Medical College, Ludhiana 2Professor & Ex-Head of Microbiology Christian Medical College, Ludhiana 3Assistant Professor Department of Biochemistry Christian Medical College, Ludhiana ABSTRACT Enzymes are produced intracellularly, and released into the plasma and body fluids, where their activities can be measured by their abilities to accelerate the particular chemical reactions they catalyze. But different serum enzymes are raised when different tissues are damaged. So serum enzyme determination can be used both to detect cellular damage and to suggest its location in situ. Some of the biochemical markers such as alanine aminotransferase, aspartate aminotransferase, alkaline phasphatase, gamma glutamyl transferase, nucleotidase, ceruloplasmin, alpha fetoprotein, amylase, lipase, creatine phosphokinase and lactate dehydrogenase are mentioned to evaluate diseases of liver, pancreas, skeletal muscle, bone, etc. Such enzyme test may assist the physician in diagnosis and treatment. KEYWORDS: Liver Function tests, Serum Amylase, Lipase, CPK and LDH. INTRODUCTION mitochondrial AST is seen in extensive tissue necrosis during myocardial infarction and also in chronic Liver diseases like liver tissue degeneration DIAGNOSTIC SERUM ENZYME and necrosis². But lesser amounts are found in Enzymes are very helpful in the diagnosis of brain, pancreas and lung. Although GPT is plentiful cardiac, hepatic, pancreatic, muscular, skeltal and in the liver and occurs only in the small amount in malignant disorders. Serum for all enzyme tests the other tissues. -
PDE6) by the Glutamic Acid- Rich Protein-2 (GARP2)
University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 2013 Regulation of the catalytic and allosteric properties of photoreceptor phosphodiesterase (PDE6) by the glutamic acid- rich protein-2 (GARP2) Wei Yao Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Yao, Wei, "Regulation of the catalytic and allosteric properties of photoreceptor phosphodiesterase (PDE6) by the glutamic acid-rich protein-2 (GARP2)" (2013). Doctoral Dissertations. 747. https://scholars.unh.edu/dissertation/747 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. REGULATION OF THE CATALYTIC AND ALLOSTERIC PROPERTIES OF PHOTORECEPTOR PHOSPHODIESTERASE (PDE6) BY THE GLUTAMIC ACID-RICH PROTEIN-2 (GARP2) BY WEI YAO B.S., Jinan University, 2007 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry September, 2013 UMI Number: 3575987 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di!ss0?t&iori Piiblist’Mlg UMI 3575987 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. -
Biased Signaling of G Protein Coupled Receptors (Gpcrs): Molecular Determinants of GPCR/Transducer Selectivity and Therapeutic Potential
Pharmacology & Therapeutics 200 (2019) 148–178 Contents lists available at ScienceDirect Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera Biased signaling of G protein coupled receptors (GPCRs): Molecular determinants of GPCR/transducer selectivity and therapeutic potential Mohammad Seyedabadi a,b, Mohammad Hossein Ghahremani c, Paul R. Albert d,⁎ a Department of Pharmacology, School of Medicine, Bushehr University of Medical Sciences, Iran b Education Development Center, Bushehr University of Medical Sciences, Iran c Department of Toxicology–Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Iran d Ottawa Hospital Research Institute, Neuroscience, University of Ottawa, Canada article info abstract Available online 8 May 2019 G protein coupled receptors (GPCRs) convey signals across membranes via interaction with G proteins. Origi- nally, an individual GPCR was thought to signal through one G protein family, comprising cognate G proteins Keywords: that mediate canonical receptor signaling. However, several deviations from canonical signaling pathways for GPCR GPCRs have been described. It is now clear that GPCRs can engage with multiple G proteins and the line between Gprotein cognate and non-cognate signaling is increasingly blurred. Furthermore, GPCRs couple to non-G protein trans- β-arrestin ducers, including β-arrestins or other scaffold proteins, to initiate additional signaling cascades. Selectivity Biased Signaling Receptor/transducer selectivity is dictated by agonist-induced receptor conformations as well as by collateral fac- Therapeutic Potential tors. In particular, ligands stabilize distinct receptor conformations to preferentially activate certain pathways, designated ‘biased signaling’. In this regard, receptor sequence alignment and mutagenesis have helped to iden- tify key receptor domains for receptor/transducer specificity.