Dodgson S J, Contino L C
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085.
Arch Biochem Biophys. 1988 Jan;260(1):334-41. doi: 10.1016/0003-9861(88)90457-2.
Mitochondrial carbonic anhydrase has previously been quantitated in liver mitochondria; it was not detected in guinea pig kidney cortical mitochondria. Evidence of this enzyme in rat kidney cortical mitochondria is reported. Electron microscopy showed that intact mitochondria were free of other intracellular organelles. When intact kidney mitochondria were added to isotonic 3'-(N'-morpholino) propanesulfonic acid buffer with 25 mM KHCO3 (1% labeled with 18O) the rate of disappearance of C18O16O was biphasic; this indicates that there is carbonic anhydrase within the inner mitochondrial membrane. Intact rat kidney mitochondria were assayed for carbonic anhydrase activity at 4 degrees C by the changing pH technique. The rate of CO2 hydration in the presence and absence of intact mitochondria was identical; this rate increased when Triton X-100 was added which indicates that all carbonic anhydrase is inside the inner mitochondrial membrane. Carbonic anhydrase activity was quantitated as kenz (units, ml.s-1 mg-1 mitochondrial protein) at 37 degrees C, pH 7.4, in 25 mM NaHCO3 (1% labeled with 18O) by following the rate of disappearance of C18O16O from solutions before and after addition of disrupted mitochondria. Values of Kenz for liver and kidney mitochondria from rats given free access to normal rat chow and water at neutral pH were 0.06 and 0.08 (respectively). Values of kenz for liver and kidney mitochondria from rats fed as above and with free access to water adjusted to pH 2.5 with HCl were 0.04 and 0.16, respectively. Values of kenz for rats starved for 48 h were 0.06 and 0.12 (respectively). The values of kenz remained 0.11-0.14 in liver mitochondria from guinea pigs fed normally, given dilute acid, or starved and the value was always at zero in guinea pig kidney mitochondria. Values of Kenz were measured with disrupted mitochondria by the 18O technique as a function of pH at 25 degrees C, 25 to 75 mM NaHCO3, ionic strength 0.3. From pH 7.0 to 8.0 kenz increased threefold for mitochondria from rat liver, fed rat kidney, and acid rat kidney, and increased eightfold for mitochondria from guinea pig liver. kenz was decreased similarly by increasing HCO3- in mitochondria from rat liver, fed kidney, and acid kidney; it is concluded that carbonic anhydrase in rat liver mitochondria is probably the same isozyme as in rat kidney mitochondria. The published observation that rat kidney cortices are up to 10 times as gluconeogenic from pyruvate as guinea pig kidney cortices can be explained by the presence of mitochondrial carbonic anhydrase in rat but not guinea pig mitochondria.
线粒体碳酸酐酶此前已在肝线粒体中进行了定量分析；在豚鼠肾皮质线粒体中未检测到该酶。本文报道了大鼠肾皮质线粒体中存在这种酶的证据。电子显微镜显示完整的线粒体没有其他细胞内细胞器。当将完整的肾线粒体添加到含有25 mM KHCO₃（1%用¹⁸O标记）的等渗3'-(N'-吗啉代)丙烷磺酸缓冲液中时，C¹⁸O¹⁶O的消失速率呈双相性；这表明线粒体内膜中存在碳酸酐酶。通过改变pH值技术在4℃下测定完整大鼠肾线粒体的碳酸酐酶活性。在有和没有完整线粒体的情况下，CO₂水合速率相同；添加Triton X - 100后该速率增加，这表明所有碳酸酐酶都在线粒体内膜内部。在37℃、pH 7.4、25 mM NaHCO₃（1%用¹⁸O标记）条件下，通过跟踪添加破碎线粒体前后溶液中C¹⁸O¹⁶O的消失速率，将碳酸酐酶活性定量为kenz（单位：ml·s⁻¹·mg⁻¹线粒体蛋白）。自由摄取正常大鼠饲料和水且处于中性pH值的大鼠的肝和肾线粒体的Kenz值分别为0.06和0.08。以同样方式喂养但自由摄取用HCl调节至pH 2.5的水的大鼠的肝和肾线粒体的kenz值分别为0.04和0.16。饥饿48小时的大鼠的kenz值分别为0.06和0.12。正常喂养、给予稀酸或饥饿的豚鼠肝线粒体的kenz值保持在0.11 - 0.14，而豚鼠肾线粒体的值始终为零。通过¹⁸O技术在25℃、25至75 mM NaHCO₃、离子强度0.3的条件下，用破碎的线粒体测量Kenz值作为pH的函数。从pH 7.0到8.0，大鼠肝、喂食大鼠肾和酸性大鼠肾的线粒体的kenz增加了三倍，豚鼠肝线粒体的kenz增加了八倍。大鼠肝、喂食肾和酸性肾的线粒体中HCO₃⁻增加时，kenz也同样降低；由此得出结论，大鼠肝线粒体中的碳酸酐酶可能与大鼠肾线粒体中的是同一种同工酶。已发表的观察结果表明，大鼠肾皮质从丙酮酸生成葡萄糖的能力是豚鼠肾皮质的10倍，这可以通过大鼠线粒体中存在线粒体碳酸酐酶而豚鼠线粒体中不存在来解释。
