Rat kidney mitochondrial carbonic anhydrase.
Rat kidney mitochondrial carbonic anhydrase.
复制标题
大鼠肾线粒体碳酸酐酶。
DOI：
10.1016/0003-9861(88)90457-2
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发表时间：
1988
影响因子：
3.9
通讯作者：
Contino,LC
中科院分区：
文献类型：
--
作者：
Dodgson,SJ;Contino,LC
线粒体碳酸酐酶先前已在肝脏线粒体中定量;在豚鼠肾皮质线粒体中未检测到。这种酶在大鼠肾皮质线粒体的证据报告。电子显微镜显示完整的线粒体没有其他细胞器。当完整的肾线粒体加入到含25 mmKHCO_3（1%~（18）O标记）的等渗3′-（N′-吗啉代）丙磺酸缓冲液中时，C_（18）O_（16）O的消失率呈双相性，这表明线粒体内膜内存在碳酸酐酶。通过改变pH技术在4 °C下测定完整大鼠肾线粒体的碳酸酐酶活性。在存在和不存在完整线粒体的情况下，CO2水合的速率是相同的;当加入Triton X-100时，该速率增加，这表明所有碳酸酐酶都在线粒体内膜内。在37 °C，pH 7.4，25 mmNaHCO 3（1%标记有18 O）中，通过跟踪加入破碎线粒体前后溶液中C18 O 16 O的消失速率，以Kenz（单位，ml · s− 1 mg − 1线粒体蛋白）定量碳酸酐酶活性。自由进食正常大鼠饲料和中性pH水的大鼠肝和肾线粒体的Kenz 0值分别为0.06和0.08。用上述方法喂养大鼠，并自由饮水（用盐酸调节pH值至2.5），其肝和肾线粒体的Kenzf值分别为0.04和0.16。饥饿48 h的Kenzz值分别为0.06和0.12。在正常喂养、稀酸和饥饿的豚鼠肝线粒体中Kenz 0值为0.11-0.14，在豚鼠肾线粒体中Kenz 0值始终为0。在25 °C、25 - 75 mmNaHCO 3、离子强度0.3条件下，通过18 O技术，用破碎的线粒体测量Kenzs值，作为pH值的函数。从pH7.0到8.0，大鼠肝脏、进食大鼠肾脏和酸性大鼠肾脏线粒体的Kenz 3增加了3倍，豚鼠肝脏线粒体的Kenz 3增加了8倍;大鼠肝脏、进食大鼠肾脏和酸性肾脏线粒体的Kenz 3随着HCO− 3的增加而减少;由此得出结论，大鼠肝脏线粒体中的碳酸酐酶可能与大鼠肾脏线粒体中的碳酸酐酶同工酶相同。已发表的观察结果表明，大鼠肾皮质的丙酮酸致炎性是豚鼠肾皮质的10倍，这可以通过大鼠而不是豚鼠线粒体中存在线粒体碳酸酐酶来解释。
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 mmKHCO3(1% labeled with18O) 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 °C by the changing pH technique. The rate of CO2hydration 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 asKenz(units, ml · s−1mg−1mitochondrial protein) at 37 °C, pH 7.4, in 25 mmNaHCO3(1% labeled with18O) by following the rate of disappearance of C18O16O from solutions before and after addition of disrupted mitochondria. Values ofKenzfor 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 ofKenzfor 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 ofKenzfor rats starved for 48 h were 0.06 and 0.12 (respectively). The values ofKenzremained 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 ofKenzwere measured with disrupted mitochondria by the18O technique as a function of pH at 25 °C, 25 to 75 mmNaHCO3, ionic strength 0.3. From pH 7.0 to 8.0Kenzincreased threefold for mitochondria from rat liver, fed rat kidney, and acid rat kidney, and increased eightfold for mitochondria from guinea pig liver.Kenzwas decreased similarly by increasing HCO−3in 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.