Isolation of a Ca2+ or Mg2+-activated ATPase (ecto-ATPase) from bovine brain synaptic membranes

Hohmann, Judit; Kowalewski, H; Vogel, Manfred; Zimmermann, Herbert

doi:10.1016/0005-2736(93)90241-q

Cited by 27 publications

(23 citation statements)

References 48 publications

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“…The ecto-ATPase has its active site exposed to the external surface of the plasma membrane (Yoshimura et al 1983, Lin and Russell 1988, Dombrowski et al 1993, Hohmann et al 1993, Smith et al 1999. In our study, the abluminal localization…”

supporting

confidence: 51%

“…Substituting Ca 2ϩ with Mg 2ϩ also identifi es the ecto-ATPase, because Ca 2ϩ -ATPase specifi cally requires Ca 2ϩ for the cytochemical reaction, while ecto-ATPase can function with any divalent ion to produce the reaction product. These are established characteristics of ecto-ATPases (Hamlyn and Senior 1983, Lin and Russell 1988, Cheung et al 1992, Hohmann et al 1993, Plesner 1995, Maxwell et al 1995, Martin-Romero et al 1996, Kegel et al 1997, Smith and Kirley 1998, Zimmermann et al 1998, Zinchuk et al 1999. All media were prepared just before use.…”

Section: Fixationmentioning

confidence: 99%

“…The negative controls have little value, however, if the cytochemical study uses the incubation medium of Ando et al (1981), because the medium co-localizes ecto-ATPase. The value of the negative control in nervous tissues is reduced further, because ecto-AT-Pase is distributed widely in nervous tissues (Kostka et al 1990, Hohmann et al 1993, James and Richardson 1993, Allen and Seyfried 1994, Maxwell et al 1995, Nitahara et al 1995, Sperlagh et al 1995, Martin-Romero et al 1996, Kegel et al 1997, Smith and Kirley 1998, Zimmermann et al 1998, Kittel 1999, Zinchuk et al 1999. Other negative controls that have been used in Ca 2+ -ATPase cytochemistry include addition of Ca 2+ chelators such as EDTA or EGTA, heating the Fig.…”

Section: Limitations Of Commonly Used Controls Not Accounting For Ectmentioning

confidence: 99%

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Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase

Manoonkitiwongsa

Whitter

Chavez

et al. 2009

Biotechnic & Histochemistry

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Ca2+-ATPase cytochemistry frequently uses the incubation medium of Ando et al. that was introduced in 1981. Some studies, however, have suggested that this medium localizes ecto-ATPase in addition to Ca2+-ATPase and that Ca2+-ATPase is sensitive to fixation. Strong activity of the enzyme on the luminal surface of the blood-brain barrier (BBB) also is considered indicative of immature or pathological microvessels. We address here five questions. 1) Is the incubation medium of Ando et al. specific for BBB Ca2+-ATPase or does it also localize ecto-ATPase? 2) How are the two enzymes distributed in the BBB? 3) How would data interpretation be prone to error if the cytochemical study does not use controls identifying ecto-ATPase? 4) Does the amount of reaction product of both enzymes vary significantly when the cortical tissue is exposed to different fixatives? 5) Does the presence of Ca2+-ATPase on the luminal membrane of the BBB necessarily indicate immature or abnormal brain endothelial cells? Adult male Sprague-Dawley rats were perfused with one of two different fixatives and vibratome slices of the brain cortex were incubated in the medium of Ando et al. The controls used were those demonstrating the ecto-ATPase and those that do not. The results indicate that the incubation medium is not specific for Ca2+-ATPase, because it also localizes the ecto-ATPase. Ca2+-ATPase appears to be localized primarily on the luminal surface of the BBB, while ecto-ATPase is localized on both the luminal and abluminal surfaces. The portion of the reaction product contributed by Ca2+-ATPase would not have been identified if the controls uniquely identifying the ecto-ATPase had not been used. The amount of reaction product formed by Ca2+-ATPase is strongly dependent on the type of fixative used. The strong localization of Ca2+-ATPase on the luminal surface of the BBB is not only normal, but also better accounts for the physiological homeostasis of Ca2+ across the blood-brain interface and should not be interpreted as indicative of immature or pathological microvessels.

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supporting

confidence: 51%

Section: Fixationmentioning

confidence: 99%

Section: Limitations Of Commonly Used Controls Not Accounting For Ectmentioning

confidence: 99%

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Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase

Manoonkitiwongsa

Whitter

Chavez

et al. 2009

Biotechnic & Histochemistry

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“…However, in the case of P2X(1) receptors, alanine substitution mutagenesis studies indicate that coordinated binding of the positive charge on magnesium-complexed ATP by negatively charged amino acids is not required for activation of the receptor by ATP (Ennion et al, 2001). Since hydrolysis of ATP by ectoATPases may be dependent on divalent cations (Nagy et al, 1986;Hohmann et al, 1993), [Mg 2ϩ ] o and [Ca 2ϩ ] o could also influence extracellular purinergic signaling in this manner. However, we found that both ATP release and calcein blue release were significantly modulated by the extracellular concentrations of Ca 2ϩ and Mg 2ϩ , with Ca 2ϩ representing a more potent modulator than Mg 2ϩ .…”

Section: Discussionmentioning

confidence: 99%

Modulation of intercellular calcium signaling in astrocytes by extracellular calcium and magnesium

Stout

Charles

2003

Glia

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The extracellular concentrations of Ca 2ϩ and Mg 2ϩ are well known to play important roles in the function of the central nervous system. We examined the effects of extracellular Ca 2ϩ and Mg 2ϩ on ATP release and intercellular signaling in astrocytes. [Ca 2ϩ ] o -activated whole cell currents consistent with currents through connexin hemichannels. These currents were inhibited by extracellular Mg 2ϩ . We conclude that extracellular divalent cations modulate intercellular Ca 2ϩ signaling in astrocytes by modulating the release of ATP, possibly via connexin hemichannels.

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“…Judging from molecular size similarities, some of our labeled polypeptides might also include ecto-ATPase subunits. For example, the 52/54-kDa bands appeared in the same molecular size range as the deglycosylated form of an ecto-ATPase from chicken gizzard [ 53 kDa (Stout and Kirley, 1994) ] and an ecto-ATPase from bovine brain [50 kDa (Hohmann et al, 1993)] . Our 82/ 84-kDa ATP-binding protein may also be related to ecto-ATPases because Rosenberg et al (1991) purified an ectonucleotide triphosphatase enzyme from chicken oviduct that has a major subunit of -80 kDa .…”

Section: Discussionmentioning

confidence: 99%

ATP‐Binding Proteins on the External Surface of Synaptic Plasma Membranes: Identification by Photoaffinity Labeling

Nagy¹,

Shuster

1995

Journal of Neurochemistry

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8‐Azidoadenosine triphosphate labeled in the α or γ position with 32P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate‐polyacrylamide gel electrophoretic patterns, UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5–10 µM resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8‐azidoadenosine [α‐32P]triphosphate‐labeled protein complex of ∼45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 mM EDTA, a band at 44 kDa was labeled within this ATP‐binding complex. The labeling pattern of the subbands of this 45/46‐kDa complex is consistent with these bands being extracellular ATP‐binding proteins on the surface of the synaptosome.

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Isolation of a Ca2+ or Mg2+-activated ATPase (ecto-ATPase) from bovine brain synaptic membranes

Cited by 27 publications

References 48 publications

Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase

Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase

Modulation of intercellular calcium signaling in astrocytes by extracellular calcium and magnesium

ATP‐Binding Proteins on the External Surface of Synaptic Plasma Membranes: Identification by Photoaffinity Labeling

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Isolation of a Ca2+ or Mg2+-activated ATPase (ecto-ATPase) from bovine brain synaptic membranes

Cited by 27 publications

References 48 publications

Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase

Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase

Modulation of intercellular calcium signaling in astrocytes by extracellular calcium and magnesium

ATP‐Binding Proteins on the External Surface of Synaptic Plasma Membranes: Identification by Photoaffinity Labeling

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Product

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Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase

Blood-brain barrier Ca²⁺-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca²⁺-specific ATPase from ecto-ATPase