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Proc Natl Acad Sci U S A
2013 Jan 22;1104:1494-9. doi: 10.1073/pnas.1221213110.
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GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons.
Schneider HP
,
Alt MD
,
Klier M
,
Spiess A
,
Andes FT
,
Waheed A
,
Sly WS
,
Becker HM
,
Deitmer JW
.
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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.
Fig. 3.
Evidence for cytosolic expression of CA IV in oocytes by immunocytochemistry (A�D) and by mass spectrometry (E and F). Fluorescent labeling of oocytes injected with CA IV�cRNA (A and C) and of native oocytes (B and D) in the confocal microscope (A and B) and in combined fluorescence and transmission mode (C and D). The same settings of the confocal microscope were applied to all images. (E) Original recordings of the degradation of 18O-labeled CO2 as measured by mass spectrometry. The first minute of the recordings shows the spontaneous degradation. The black arrow indicates the addition of oocyte lysate, prepared from 20 cells expressing CA IV�WT (blue) and CA IV�V165Y (orange), respectively, and native oocytes (black). (F) CA activity as measured by mass spectrometry in intact and lysed oocytes expressing CA IV or injected with CA II. For each experiment, 100 intact or 20 lysed oocytes were added to the measuring cuvettete. Catalytic activity was then calculated as activity per single oocyte. (G) Calibration curve for quantification of CA IV in Xenopus oocytes. Catalytic activity of defined amounts of CA IV protein was measured by mass spectrometry and fitted by linear regression to calculate the amount of expressed CA IV. (H) Absolute amount of extra- and intracellular CA IV concentration as calculated from the catalytic activity measured in intact and lysed oocytes, respectively, and the calibrated activity curve.
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