Koncsos Gábor, Varga Zoltán V, Baranyai Tamás, Ferdinandy Péter, Schulz Rainer, Giricz Zoltán, Boengler Kerstin
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089 Budapest, Hungary.
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089 Budapest, Hungary; Pharmahungary Group, Szeged, Hungary.
J Pharmacol Toxicol Methods. 2018 May-Jun;91:50-58. doi: 10.1016/j.vascn.2018.01.004. Epub 2018 Jan 31.
In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins.
SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot.
MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM.
Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.
在心脏中，肌膜下（SSM）、肌原纤维间（IFM）和核周线粒体代表线粒体的三种亚型。IFM分离过程中最常用的蛋白酶是蛇毒蛋白酶，然而，其对线粒体蛋白检测的影响仍不清楚。因此，我们研究了蛇毒蛋白酶处理是否会影响线粒体蛋白的定量。
从小鼠和大鼠心脏中分离SSM和IFM。在IFM分离过程中，蛇毒蛋白酶活性通过离心停止（常规方案，IFM+N）或通过苯甲基磺酰氟（PMSF，IFM+N+I）抑制。通过蛋白质印迹法测定位于不同线粒体区室（外膜：线粒体融合蛋白1（MFN1）和2（MFN2）；膜间隙：p66shc；内膜（连接蛋白43（Cx43）））的蛋白质以及蛋白质去糖基化酶DJ-1的量。
MFN2和Cx43主要存在于从小鼠和大鼠心脏分离的SSM中。MFN1和p66shc在SSM和IFM+N中的含量相似，而与SSM相比，DJ-1在IFM+N中的水平更高。在来自小鼠的IFM+N+I样品中，MFN2的量增加，但Cx43的量没有增加。蛇毒蛋白酶或PMSF对蛇毒蛋白酶的抑制对SSM或IFM的氧消耗没有影响。
虽然常规方案表明MFN2主要定位于SSM，但PMSF对蛇毒蛋白酶的抑制使IFM中MFN2的信号增加到与SSM中的信号相同，表明IFM中MFN2被低估。因此，在使用基于蛇毒蛋白酶的分离方法评估线粒体蛋白分布时，应考虑蛋白酶敏感性。
