Osicka T M, Comper W D
Biochemistry Department, Monash University, Clayton, Victoria, Australia.
Kidney Int. 1995 Jun;47(6):1630-7. doi: 10.1038/ki.1995.227.
Studies in isolated perfused rat kidney have demonstrated that it exhibits apparently normal charge selectivity and tubular uptake of anionic horseradish peroxidase (aHRP; pI < 4.0) and neutral horseradish peroxidase (nHRP; pI = 7.5) when these proteins are measured for their enzyme activity. The absolute fractional clearance values for aHRP and nHRP were 0.006 +/- 0.002 and 0.041 +/- 0.007, respectively. It is evident, however, that the enzyme assay for horseradish peroxidase severely underestimates the quantity of protein in urine as compared to measurement of its tritium labeled form through radioactivity. Fractional clearances estimated by radioactivity and corrected for tubular reabsorption for [3H]aHRP and [3H]nHRP were 0.040 +/- 0.029 and 0.099 +/- 0.043, respectively, compared to those estimated by enzyme activity which were 0.012 +/- 0.004 and 0.070 +/- 0.037, respectively. While charge selectivity between the anionic and neutral forms of HRP was still evident, albeit significantly reduced, the major feature of this type of analysis is that the clearance of the aHRP protein is significantly increased compared to that determined by enzyme assay. This difference correlates with the observation that the aHRP protein is markedly degraded (61 to 65%), as determined by gel chromatography, during filtration. Similar degradation was seen in urine fractions collected after the aHRP protein was administered in vivo. Degradation also occurred for the nHRP protein in both the perfused kidney and in vivo but to a far lesser extent (approximately 14 to 21%). These studies demonstrate that the anionic form of HRP was preferentially degraded during filtration and that charge selectivity for differently charged proteins is not as marked as originally thought.
对离体灌注大鼠肾脏的研究表明，当检测阴离子型辣根过氧化物酶（aHRP；pI < 4.0）和中性辣根过氧化物酶（nHRP；pI = 7.5）的酶活性时，肾脏表现出明显正常的电荷选择性和对这些蛋白质的肾小管摄取。aHRP和nHRP的绝对分数清除率值分别为0.006±0.002和0.041±0.007。然而，很明显，与通过放射性测量其氚标记形式相比，辣根过氧化物酶的酶测定法严重低估了尿液中的蛋白质量。通过放射性估计并经肾小管重吸收校正的[3H]aHRP和[3H]nHRP的分数清除率分别为0.040±0.029和0.099±0.043，而通过酶活性估计的分数清除率分别为0.012±0.004和0.070±0.037。虽然HRP阴离子和中性形式之间的电荷选择性仍然明显，尽管显著降低，但这种分析类型的主要特征是，与酶测定法确定的相比，aHRP蛋白的清除率显著增加。这种差异与以下观察结果相关：通过凝胶色谱法测定，aHRP蛋白在过滤过程中明显降解（61%至65%）。在体内给予aHRP蛋白后收集的尿样中也观察到类似的降解。灌注肾脏和体内的nHRP蛋白也发生了降解，但程度要小得多（约14%至21%）。这些研究表明，HRP的阴离子形式在过滤过程中优先降解，并且对不同电荷蛋白质的电荷选择性不如最初认为的那么明显。
