Expression and Characterization of Chicken Muscle Ecto-ATPase in Mammalian COS Cells

Kirley, Terence L.; Gerber, Lamar K.; Smith, Thomas M.

doi:10.1080/152165499307431

Cited by 4 publications

(4 citation statements)

References 14 publications

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“…The majority of the enzymes hydrolysing ATP prefer Mg 2+ –ATP complex as a substrate [3]. NTPDase2 derived from chicken gizzard smooth muscle was activated the most strongly by Mg 2+ [36], whereas NTPDase1 from human placenta most efficiently hydrolysed ATP in the presence of Ca 2+ [37]. Our results indicate that the complexes Mg 2+ –ATP or Mn 2+ –ATP are preferred as substrate by synaptosomal NTPDases, while in the case of ADP, the Ca 2+ –ADP complexes are hydrolysed most effectively.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Kukulski

Komoszyński

2003

European Journal of Biochemistry

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We purified to homogeneity and characterized NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. SDS/PAGE and immunoblotting with antibodies specific to these enzymes revealed a molecular mass estimated at 72 kDa for NTPDase1 and 66 for NTPDase2. Both enzymes exhibited kinetic properties typical for all members of the NTPDase family, e.g. low substrate specificity for triand diphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. The calculated K m value for NTPDase1 in respect to ATP as a substrate (97 lM) was three times lower in comparison to analogous values for NTPDase2 (270 lM). Additionally, NTPDase1 had a three times higher K cat /K m coefficient than NTPDase2 (860 and 833 lmol productAEs )1 , respectively). We have also demonstrated that in spite of differences in the affinity of ATP for both hydrolases, these enzymes have similar molecular activity. Taken together, these results indicate that NTPDase1 would terminate P2 receptor-mediated signal transmission whereas activity of NTPDase2 may contribute to decreasing high (toxic) concentrations of ATP and/or to production of another signal molecule, ADP.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Kukulski

Komoszyński

2003

European Journal of Biochemistry

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“…Native gel electrophoresis of COS cell-expressed chicken (20) and human ecto-ATPases (eNTPDase2) revealed that both enzymes had virtually the same mobility on a 6% acrylamide native gel, as judged by gel blot detection of the proteins after transfer to PVDF membrane (data not shown). However, because mobility in native gels is determined both by charge and size, we also determined the apparent size of the digitonin-solubilized ecto-ATPase by size-exclusion gel chromatography.…”

Section: Oligomeric Size Analysis Of Digitonin-solubilized Entpdasesmentioning

confidence: 97%

“…Transfected COS cell membranes (or rabbit skeletal transverse tubule membranes containing ecto-ATPase [20]) at 0.1 mg/ mL total protein were solubilized by adding digitonin to a nal concentration of 1% and mixing for 10 min at room temperature. The insoluble proteins were separated by centrifugation and the soluble proteins were applied to either a native Laemmli gel (no SDS was used, but the gel contained 0.1% digitonin; see [6]) or to a Sephacryl S-300 column equilibrated in a solution of 20 mM MOPS and 5 mM MgCl 2 , pH 7.4, containing 0.1% digitonin.…”

Section: Solubilization Native Gel Electrophoresis and Size-exclusimentioning

confidence: 99%

Expression and Characterization of Human Ecto‐ATPase and Chimeras with CD39 Ecto‐Apyrase

Hicks-Berger

Kirley

2000

IUBMB Life

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Human ecto-ATPase (ecto-nucleoside triphosphate diphosphohydrolase 2 [eNTPDase2], also known as CD39L1) has been expressed and characterized in COS cells. It exhibits some unusual enzymology that is similar to a few members of this class of proteins but different from the majority of the family members. Hydrolysis of ATP by human ecto-ATPase is nonlinear with time, and its activity is stimulated/stabilized by both the lectin concanavalin A and the chemical cross-linking agent disuccinimidyl suberate. Like other members of the eNTPDase family, the human ecto-ATPase is a tetramer, the activity of which depends on its glycosylation. Chimeras of this protein with human CD39 (eNTPDasel) were constructed to test the hypothesis that the N-terminal half of these proteins regulates nucleotide specificity. The two chimeras generated demonstrated that the N-terminal half of these proteins is crucial for determining the relative activities of the nucleoside di- and triphosphatases. Chemical cross-linking of the two chimeras suggests that disuccinimidyl suberate interacts with the C-terminal half of ecto-ATPase in a manner that results in an increase of activity for both the ecto-ATPase and the ecto-apyrase/ecto-ATPase chimera.

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“…Stimulation of the avian enzyme by lectin was shown to occur through direct binding of the lectin to oligosaccharide residues, which might form part of the ecto-ATPase structure, because after desialylation no activation with wheatgerm agglutinin was observed and α-methylmannoside prevented stimulation by Con A [5,6]. Furthermore, glycosylation is crucial for chicken muscle ecto-ATPase, because expression of the enzyme in mammalian cells in the presence of tunicamycin resulted in an inactive, unfolded protein [31].…”

Section: Oligomeric Statementioning

confidence: 99%

Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle

Megías

Martinez-Senac

Delgado

et al. 2001

Biochemical Journal

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Transverse tubule (T-tubule) ecto-ATPase from chicken skeletal muscle is an integral membrane glycoprotein that seems to exist as a homodimer and exhibits unusual properties. Treatment of T-tubule membranes with concanavalin A (Con A) did not significantly affect the thermal variation of the fluorescence anisotropy of vesicles labelled with 1,6-diphenyl-1,3,5-hexatriene or trimethylammonium-1,6-diphenyl-1,3,5-hexatriene. Cross-linking of membrane components with glutaraldehyde elicited effects on ecto-ATPase activity very similar to those of Con A treatment: a severalfold increase in activity, a decrease in Triton X-100 sensitivity and a requirement to be present before ATP to exert its action. In addition, glutaraldehyde and Con A normalized the temperature dependence and the kinetic behaviour of the enzyme. Membrane-perturbing agents (detergents, alcohols and cholesterol oxidase), with the sole exception of digitonin, caused a marked decrease in ecto-ATPase activity; the prior presence of Con A prevented this inhibition, whereas when the lectin was added after the membrane perturbing agent, recovery of the activity was not always possible. The addition of nucleotides before Con A led to a suppression of ecto-ATPase stimulation; it occurred when the nucleotide was hydrolysed (ATP or UTP) and when it was not (adenosine 5′-[β,γ-imido]triphosphate) and even in the presence of 3mM Pi. A model is proposed for the complex regulatory mechanisms of chicken T-tubule ecto-ATPase that involves the occurrence of two different catalytic states in an equilibrium modulated by lectins and cross-linking agents, by the structure of the membrane and by the presence of ligands for a regulatory site.

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Expression and Characterization of Chicken Muscle Ecto-ATPase in Mammalian COS Cells

Cited by 4 publications

References 14 publications

Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Expression and Characterization of Human Ecto‐ATPase and Chimeras with CD39 Ecto‐Apyrase

Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle

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