Silverman D N, Tu C, Wynns G C
J Biol Chem. 1976 Jul 25;251(14):4428-35.
The depletion of 18O from CO2, caused by the exchange of oxygen between CO2 and water during the hydration-dehydration cycle, is catalyzed by carbonic anhydrase. This depletion process at chemical equilibrium in the presence of erythrocytes is biphasic, exhibiting a very rapid depletion rate immediately following the addition of cells to an isotonic solution containing 18O-enriched CO2, followed by a much slower depletion rate. It is hypothesized that these depletion characteristics are caused by the diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly due to the large intracellular carbonic anhydrase content. Kinetic equations which describe this hypothesis are solved and a rate constant is obtained which represents the depletion of 18O in CO2 caused by the presence of red cells. These are equilibrium experiments with no net uptake or loss of CO2 in the cells. Consequently, depletion processes are not limited in rate by bicarbonate-chloride exchange or proton distribution across the membrane. The purpose of these measurements is to determine whether the rate of 18O depletion in red cell suspensions is determined by carbonic anhydrase activity in the cell or by the diffusion process by which CO2 enters the cell. This goal is achieved by partially inhibiting carbonic anhydrase with acetazolamide. The rate constant representing 18O depletion caused by the presence of red cells is unchanged, even though up to 90% of carbonic anhydrase is inhibited. From this rate constant the permeability constant of the membrane of rat erythrocytes to CO2 at 25 degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to block HCO3 -flux across the membrane. Using no picrate and allowing HCO3-flux to introduce an error in the measurements, the permeability constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants measured by this technique include the diffusion barrier to CO2 not only of the red cell membrane but also of a portion of the intracellular medium.
在水合-脱水循环过程中，二氧化碳与水之间的氧交换导致二氧化碳中18O的消耗，这一过程由碳酸酐酶催化。在红细胞存在的情况下，处于化学平衡状态的这种消耗过程是双相的，在将细胞加入含有富集18O的二氧化碳的等渗溶液后，立即呈现出非常快速的消耗速率，随后消耗速率则慢得多。据推测，这些消耗特征是由标记的二氧化碳扩散到红细胞中所致，在红细胞内，由于细胞内碳酸酐酶含量高，消耗迅速发生。求解了描述这一假设的动力学方程，并得到了一个速率常数，该常数表示红细胞的存在导致二氧化碳中18O的消耗。这些是平衡实验，细胞内没有二氧化碳的净摄取或损失。因此，消耗过程的速率不受碳酸氢盐-氯离子交换或质子跨膜分布的限制。这些测量的目的是确定红细胞悬液中18O的消耗速率是由细胞内的碳酸酐酶活性还是由二氧化碳进入细胞的扩散过程决定的。这一目标通过用乙酰唑胺部分抑制碳酸酐酶来实现。即使高达90%的碳酸酐酶被抑制，代表红细胞存在导致18O消耗的速率常数也没有变化。根据这个速率常数，在存在3.2 mM苦味酸盐（一种旨在阻止HCO3-跨膜通量的被动阴离子扩散抑制剂）的情况下，大鼠红细胞膜在25℃和pH 7.4时对二氧化碳的渗透常数被确定为(7.6±1.2)×10(-3) cm s-1。在不使用苦味酸盐并允许HCO3-通量在测量中引入误差的情况下，渗透常数为(1.6±0.4)×10(-2) cm s-1。用这种技术测量的渗透常数不仅包括红细胞膜对二氧化碳的扩散屏障，还包括细胞内部分介质的扩散屏障。
