Department of Physiology, Semmelweis University, Faculty of Medicine, Tűzoltó utca 37-47, H-1094, Budapest, Hungary.
Department of Physiology, Semmelweis University, Faculty of Medicine, Tűzoltó utca 37-47, H-1094, Budapest, Hungary.
Redox Biol. 2022 Aug;54:102385. doi: 10.1016/j.redox.2022.102385. Epub 2022 Jun 30.
Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.
过氧化物酶 4（PXDN）参与了 IV 型胶原的交联反应，IV 型胶原是基底膜的主要成分。IV 型胶原或 PXDN 的遗传改变导致基底膜完整性的破坏，可能导致发育缺陷和多种病理。因此，研究 PXDN 在（病理）生理环境中的活性具有重要意义。到目前为止，已经从纯化蛋白、细胞裂解物和去细胞化细胞外基质中报道了 PXDN 活性的测量。在这里，我们首次使用信号放大修饰的 Amplex Red 测定法报告了在活细胞中测量 PXDN 活性。我们观察到溴化物的添加增强了获得的信号，这很可能是由于 HOBr 的形成。HOBr 清除剂肌肽对信号放大的阻断支持了这一假设。药理学抑制和互补的遗传方法都证实了所获得的信号确实与 PXDN 的活性有关。我们通过研究 Brefeldin A 的作用来验证改良的测定法，以抑制分泌途径，从而阻止 PXDN 进入细胞外的 Amplex Red 染料。我们的方法为研究 PXDN 在（病理）生理环境中的活性开辟了新的可能性。
