Cao G, Cutler R G
Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institute of Health, Baltimore, Maryland 21224, USA.
Arch Biochem Biophys. 1995 Jun 20;320(1):195-201. doi: 10.1006/abbi.1995.1359.
A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent decrease in the activities of enzymes, especially of alkaline proteases, which preferentially degrade oxidatively modified protein. Recently, this hypothesis was strongly supported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhydrazine procedure described in the PBN study. We now focus on the alkaline protease activity assay and show that alkaline protease activity cannot be reliably measured in crude tissue extracts by using the fluorescamine procedure also described in the PBN study. We were, however, able to reliably measure a protease activity in crude tissue extracts at alkaline pH by using a synthetic fluorogenic peptide substrate, but no effect of aging or PBN treatment was found on the protease activity in rat brain cortexes. Thus, the reported age-dependent changes in protein carbonyl formation and alkaline protease activity remain to be confirmed.
目前一种解释衰老过程的假说认为，动物组织中氧化蛋白质的积累与衰老有关。这主要基于一系列报告，这些报告显示蛋白质羰基含量随年龄增长而增加，酶活性尤其是碱性蛋白酶的活性随年龄增长而降低，碱性蛋白酶优先降解氧化修饰的蛋白质。最近，自旋捕获化合物N-叔丁基-α-苯基硝酮（PBN）具有逆转这些随年龄变化的作用这一新型效应的报告，有力地支持了这一假说。然而，我们发现，使用PBN研究中描述的2,4-二硝基苯肼法无法在组织中可靠地测量活性蛋白质羰基。我们现在关注碱性蛋白酶活性测定，并表明使用PBN研究中也描述的荧光胺法无法在粗组织提取物中可靠地测量碱性蛋白酶活性。然而，我们能够通过使用合成荧光肽底物在碱性pH条件下可靠地测量粗组织提取物中的蛋白酶活性，但未发现衰老或PBN处理对大鼠脑皮质中的蛋白酶活性有影响。因此，所报道的蛋白质羰基形成和碱性蛋白酶活性随年龄的变化仍有待证实。
