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.