首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons
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GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons

机译:GPI锚定的碳酸酐酶IV在注射cRNA的卵母细胞和小鼠神经元中均显示细胞内和细胞外活性

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摘要

Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms-such as isoforms IV, IX, XII, XIV, and XV-also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H~+ at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H~+ shifts of granule cells following CO_2 removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H~+ dynamics in the cytosol, both in injected oocytes and in mouse neurons.
机译:众所周知,可溶性胞质碳酸酐酶(CAs)参与哺乳动物细胞的细胞质的pH调节。膜结合的CA同工型,例如同工型IV,IX,XII,XIV和XV,也催化二氧化碳可逆转化为质子和碳酸氢根,但在细胞膜的细胞外表面。当人类CA同工型IV在非洲爪蟾卵母细胞中异源表达时,我们通过用离子选择微电极测量细胞膜外表面和胞质溶胶中的H〜+,不仅观察到细胞外催化CA活性,而且观察到了健壮的细胞内活性。通过免疫细胞化学证实了卵母细胞中CA IV的表达,并且通过质谱法测定了CA IV的活性。 CA IV的细胞外和细胞内催化活性可以使用相对较慢的膜渗透性的CA抑制剂苯甲酰胺进行药理分析。在缺乏CA IV的突变小鼠的急性小脑切片中,CO 2去除/添加后颗粒细胞的胞质H +迁移明显慢于野生型小鼠。我们的研究结果表明,膜相关的CA IV在细胞内贡献了强大的催化活性,并且该活性参与调节了注入卵母细胞和小鼠神经元中胞质溶胶中的H〜+动态。

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  • 作者单位

    Abteilung fuer Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Abteilung fuer Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Abteilung fuer Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany,Arbeitsgruppe Zoologie/Membrantransport, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Abteilung fuer Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Arbeitsgruppe Zoologie/Membrantransport, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104;

    Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104;

    Arbeitsgruppe Zoologie/Membrantransport, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

    Abteilung fuer Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    extracellular carbonic anhydrase; membrane surface H~+; ethoxzolamide; cerebellar granule cells; intracellular transport vesicles;

    机译:细胞外碳酸酐酶;膜表面H〜+;乙氧唑胺;小脑颗粒细胞细胞内运输小泡;
  • 入库时间 2022-08-18 00:39:53

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