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Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

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Abstract

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca2+-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with Km for Ca ions of 0.4 μM and Vmax=12.5±2.4 μmol Pi.mg−1h−1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive3H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with Ki=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >Zn≥Co>Ni. In contrast to low affinity (high Km) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 μM) and N-ethylmaleinimide (100 μM). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.

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Abbreviations

BSA:

bovine serum albumin

HA Ca-ATPase:

high-affinity Ca2+-ATPase

K-pNPPase:

onabain-sensitive, K+-stimulated p-nitrophenyl phosphatase

NEM:

N-ethylmaleinimide

SIM:

250 mM sucrose, 10 mM imidazole-HCl pH 7.4

TRIS:

tris (hydroxymethyl) aminomethane

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Author notes

  1. Peter Kostka

    Present address: Division of Biochemistry, Walter Reed Army Institute of Research, 20307-5100, Washington, D.C.

Authors and Affiliations

  1. Division of Physiology and Pharmacology, Department of Biomedical Sciences, McMaster University, L8N 3Z5, Hamilton, Ontario, Canada

    Peter Kostka, William H. Barnett & Chiu-Yin Kwan

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  1. Peter Kostka
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  2. William H. Barnett
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  3. Chiu-Yin Kwan
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Kostka, P., Barnett, W.H. & Kwan, CY. Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves. Neurochem Res 15, 833–841 (1990). https://doi.org/10.1007/BF00968562

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  • DOI: https://doi.org/10.1007/BF00968562

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